Delivery of immunoglobulin G antibodies to the rat nervous system following intranasal administration: Distribution, dose-response, and mechanisms of delivery

Niyanta N Kumar; Jeffrey J Lochhead; Michelle E Pizzo; Geetika Nehra; Sam Boroumand; Gretchen Greene; Robert G Thorne

doi:10.1016/j.jconrel.2018.08.006

. Author manuscript; available in PMC: 2020 May 18.

Published in final edited form as: J Control Release. 2018 Aug 4;286:467–484. doi: 10.1016/j.jconrel.2018.08.006

Delivery of immunoglobulin G antibodies to the rat nervous system following intranasal administration: Distribution, dose-response, and mechanisms of delivery

Niyanta N Kumar ^a, Jeffrey J Lochhead ^a, Michelle E Pizzo ^a,^b, Geetika Nehra ^a, Sam Boroumand ^a, Gretchen Greene ^a, Robert G Thorne ^a,^b,^c,^d,^*

PMCID: PMC7234798 NIHMSID: NIHMS1504903 PMID: 30081144

Abstract

The intranasal route has been hypothesized to circumvent the blood-brain and blood-cerebrospinal fluid barriers, allowing entry into the brain via extracellular pathways along olfactory and trigeminal nerves and the perivascular spaces (PVS) of cerebral blood vessels. We investigated the potential of the intranasal route to noninvasively deliver antibodies to the brain 30 min following administration by characterizing distribution, dose-response, and mechanisms of antibody transport to and within the brain after administering non-targeted radiolabeled or fluorescently-labeled full length immunoglobulin G (IgG) to normal adult female rats. Intranasal [¹²⁵I]-IgG consistently yielded highest concentrations in the olfactory bulbs, trigeminal nerves, and leptomeningeal blood vessels with their associated PVS. Intranasal delivery also resulted in significantly higher [¹²⁵I]-IgG concentrations in the CNS than systemic (intra-arterial) delivery for doses producing similar endpoint blood concentrations. Importantly, CNS targeting significantly increased with increasing dose only with intranasal administration, yielding brain concentrations that ranged from the low-to-mid picomolar range with tracer dosing (50 μg) up to the low nanomolar range at higher doses (1 mg and 2.5 mg). Finally, intranasal pre-treatment with a previously identified nasal permeation enhancer, matrix metalloproteinase-9, significantly improved intranasal [¹²⁵I]-IgG delivery to multiple brain regions and further allowed us to elucidate IgG transport pathways extending from the nasal epithelia into the brain using fluorescence microscopy. The results show that it may be feasible to achieve therapeutic levels of IgG in the CNS, particularly at higher intranasal doses, and clarify the likely cranial nerve and perivascular distribution pathways taken by antibodies to reach the brain from the nasal mucosae.

Keywords: Intranasal, Perivascular, Antibodies, Brain, Olfactory, Trigeminal

1. Introduction

Antibody-based therapeutics have gained significant momentum as potential treatments for several central nervous system (CNS) disorders, including stroke [1], Alzheimer's disease (AD) [2], Parkinson's disease (PD) [3], brain cancer [4], and multiple sclerosis [5], among others. However, drug delivery to the CNS for antibodies and other macro-molecules has thus far proven challenging [6], due in large part to the blood-brain barrier (BBB) [7] and blood-cerebrospinal fluid barriers (BCSFBs) [8, 9] that greatly restrict transport from the systemic circulation into the CNS.

Several key questions remain regarding whether and how much systemic immunoglobulin G (IgG) accesses brain parenchyma and/or CSF and the precise pathways involved. It has long been thought that circulating endogenous IgG is potentially capable of entering the CNS from the systemic circulation [10, 11], e.g., via sites such as the circumventricular organs where the BBB is absent [12, 13], but the capacity and efficiency of such pathways for IgG brain entry have remained largely unknown. Similarly, reports on the degree to which systemically administered exogenous IgG may access the brain and/or CSF have varied widely [14–18]. It is likely that many studies reporting IgG brain entry from the systemic circulation have overestimated the fraction actually present within the neuropil; indeed, recent work suggests the majority of systemically derived IgG in brain samples is sequestered within the endothelial cell compartment [17]. In light of these issues, there has been a clear need for minimally invasive techniques capable of bypassing the BBB and delivering IgG to the CNS.

Intranasal administration has received increasing attention as a potential non-invasive method capable of delivering therapeutically relevant concentrations of many different substances, including large biologics, into the CNS of rodents, monkeys, and even humans [19–22]. The intranasal route provides many potential advantages over other routes of administration: easy self-administration and dose adjustment, rapid onset of effects, avoidance of hepatic first pass elimination, and potential direct pathways to the CNS that bypass the BBB [19, 20]. Transport from the nasal mucosae to the brains of both rats and non-human primates has been suggested to occur via direct extracellular pathways along components of olfactory and trigeminal nerves [23, 24], with subsequent widespread distribution to other CNS areas via convection or dispersion within the perivascular spaces of cerebral blood vessels [25, 26].

We hypothesized that intranasal delivery may potentially be used to target antibodies as large as 150 kDa full length IgG to the CNS and, further, that antibody transport across the nasal epithelia and subsequent access to the perivascular spaces of cerebral blood vessels can be defined and manipulated for better efficiency. Reports exist suggesting that intranasal administration of specific full length IgG anti-bodies [27–29], as well as smaller antibody fragments [30], may potentially result in central delivery and responses in rodent models of AD. However, detailed descriptions of CNS IgG distribution resulting from intranasal administration, possible delivery pathways and mechanisms responsible for IgG transport from the nasal epithelia to the CNS, and strategies that might be utilized to optimize CNS delivery of intranasally applied IgG have yet to be provided. Here, we address these gaps, providing critical, new insights into the use of the non-invasive intranasal route of administration to deliver IgG to the CNS in normal rats using complementary radiometric and fluorescence-based methods.

2. Methods

2.1. Experimental design and statistical analysis

Our experimental strategy to characterize intranasal delivery of antibodies to the CNS involved (i) quantitative assessment of antibody distribution in the CNS, (ii) use of vascular control experiments to facilitate interpretation, and (iii) high resolution fluorescence imaging to better elucidate the pathways taken by antibodies to reach the brain. For quantitative assessment of antibody distribution in the CNS, we utilized highly sensitive methods of measurement for [¹²⁵I]-labeled antibodies, similar to previous studies investigating intranasal delivery of other [¹²⁵I]-labeled proteins to the CNS [23, 24]. Vascular controls were necessary to confirm intranasally applied antibodies reached the CNS at least in part by bypassing the BBB. Intranasally administered substances may either enter the systemic circulation and reach the brain by crossing the BBB and BCSFBs or access direct extracellular pathways to the CNS that bypass the BBB and BCSFBs [19, 20, 31]. Control experiments with intra-arterially administered antibodies were matched to achieve similar endpoint blood concentrations as that obtained following intranasal experiments, allowing us to interpret any differences in CNS levels between the two routes as direct targeting unique to intranasal administration [23, 25, 32]. Lower resolution ex vivo fluorescence imaging and higher resolution confocal imaging of intranasally delivered fluorophore-conjugated antibodies in the nasal passages, trigeminal nerves, and brain sections allowed us to identify the specific anatomical compartments and pathways accessed by anti-bodies to reach the brain.

All data was analyzed using SigmaPlot software (version 11.0, Systat Software). To assess significant differences between two groups, a t-test was used when data followed a normal distribution or a Mann-Whitney rank sum test was used when data did not follow a normal distribution. To assess significant differences between three or more groups, a one-way ANOVA and post hoc Bonferroni t-test were used when data followed a normal distribution or a Kruskal-Wallis one-way ANOVA on ranks and post hoc Dunn's test were used when the data did not follow a normal distribution. Data are presented as mean ± standard error (SEM). A p value of < 0.05 was accepted as statistically significant.

2.2. Animals and surgeries

All animal experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Wisconsin-Madison and were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals (8th edition, [33]). Adult female Sprague-Dawley rats (180–200 g; Envigo) were used for all in vivo experiments. Rats were housed under a 12 h light-dark cycle and fed ad libitum. Body temperature was maintained at 37 °C with a homeothermic blanket (Harvard Apparatus). Rats were anesthetized with an intraperitoneal injection of urethane (1.2 g/kg, with supplements to effect) and cannulated (20G × 1.25 in.; Exel International) through the abdominal aorta for tracer administration (for intra-arterial dosing experiments), plasma sampling, and upper body perfusion.

For in vivo experiments where cerebrospinal fluid (CSF) was serially sampled during and following intranasal or intra-arterial delivery, a cannula was surgically inserted into the cisterna magna to allow CSF sampling, as described previously [25, 34]. Briefly, rats were anesthetized and placed in a stereotaxic frame (Stoelting) in the flat skull position. Lidocaine hydrochloride 2% (0.05ml) was injected subcutaneously on the animal's scalp as an additional local anesthetic. Body temperature was maintained at 37 °C throughout the experiment using a homeothermic blanket system/rectal temperature probe with feedback control (Harvard Apparatus). The cisterna magna was exposed for cannula insertion by a dorsal midline neck incision, resection of subcutaneous tissues and muscles, and careful removal of the atlanto-occipital membrane overlying the dura. A custom cannula typically consisting of 33GA polyetheretherketone tubing (PEEK; Plastics One) cut to a length of approximately 1cm was connected via PE-10 tubing (Solomon Scientific). The PE-10 tubing was connected to an HPLC two-way shut off valve (Idex Health and Science) using tubing sleeves (Idex Health Science) and the length of tubing was kept short (~1in) to minimze dead volume. Next the HPLC two-way shut off valve was connected to a Hamilton syringe controlled by an infusion pump (Quintessential Stereotaxic Injector, Stoelting) with ~4ft of PE-10 tubing. The system was flushed with approximately 2 ml of 0.25% bovine serum albumin solution in 0.9% sterile physiological saline, pH 7.4, to coat the tubing and minimize IgG adherence to the tubing [35]; the system was then filled with 0.9% sterile physiological saline, pH 7.4, prior to insertion. A small air bubble was drawn up through the PEEK, HPLC valve, and PE-10 tubing prior to insertion to prevent intermixing between withdrawn CSF and saline in the PE-10 tubing. The PEEK cannula was advanced 1mm into the cisterna magna at a 30-de-gree angle from the horizontal following creation of a small hole in the dura with a 30GA dental needle (Exel International); the cannula was then sealed to the dura and fixed in place by cyanoacrylate.

2.3. Intranasally and intra-arterially administered molecules

2.3.1. Radiolabeled antibodies

All radioactive material was handled behind lead impregnated plexiglass shielding in a chemical fume hood and stored in lead lined containers. While handling radioactive material, appropriate PPE was worn at all times, and the work area and personnel radioactivity exposure was monitored using wipe tests, breathing zone monitors, thyroid scans post-radiolabeling, and a wearable dosimeter. Non-targeted unlabeled ChromPure rat IgG (Jackson Immunoresearch; catalog number: 012–000-003) was radiolabeled in-house with 1–2 mCi Na¹²⁵I using the Chizzonite indirect iodination method [36] with Pierce iodination tubes (Thermo Fisher Scientific). All steps, unless mentioned otherwise, were performed at room temperature. Briefly, 1ml of Tris iodination buffer (25 mM Tris HCl, 0.4 M NaCl, pH 7.5) was added to a Pierce pre-coated iodination tube to wet the oxidizing agent at the bottom of the tube. The 1 ml of Tris iodination buffer was decanted while avoiding touching the bottom of the tube. Next, 100 μl of the Tris iodination buffer was added to the same pre-coated, wet, Pierce iodination tube. 10 μL of Na¹²⁵I salt solution (1 mCi; Perkin Elmer) was added to the iodination tube and mixed with the Tris iodination buffer by gently shaking the tube. The Na¹²⁵I was allowed to react with the oxidizing agent in the Pierce iodination tube for 6 min, while gently shaking the tube every 2 min. The iodide solution was then added to a 2 ml microcentrifuge tube containing 0.5 nmoles of unlabeled Chrom-Pure rat IgG (Jackson Immunoresearch; catalog number: 012–000-003) in Tris iodination buffer. The iodide was allowed to react with the IgG for 9 min, by gently shaking the tube every 3 min. Unreacted iodide was then quenched by adding 50 μl of scavenging buffer (10 mg tyrosine/ml in Tris Iodination Buffer) and incubating for 5 min. 1 ml of Tris BSA buffer (Tris iodination buffer with 5 mM EDTA, 0.25% BSA, 0.05% sodium azide) was then added to the radiolabeled IgG solution. This radiolabeled IgG solution was then added to a 10 ml Zeba desalting spin column (7000 MW cut-off; Thermo Fisher) which had been equilibrated with 20 ml Tris/BSA Buffer. The Zeba desalting spin column was spun at 1000 ×g for 2 min at 4 °C to separate the free iodine from the labeled protein. After the spin, three 5 μl aliquots were collected to measure radioactivity by gamma counting. Gamma counting was performed using a Packard Cobra II auto Gamma counter (Perkin Elmer). The [¹²⁵I]-IgG solution was concentrated and free iodine further removed with a subsequent centrifugation using a 2 ml Amicon Ultra 100 kDa molecular weight cut-off centrifugal filter (Millipore Sigma). 2 ml of the [¹²⁵I]-IgG was added to the Amicon filter and spun at 7197 ×g (max speed) for 1 h at 4 °C using an Eppendorf 5430R centrifuge with a F-35–6-30 rotor. The filtrate was discarded in a radioactive fluid waste container and a recovery spin was performed at 1000 ×g for 2 min at 4 °C. After the spin, three 5 μl aliquots of the radiolabeled protein (diluted 1:100 in Tris BSA buffer) were collected to measure radioactivity by gamma counting. Radioactivity measurements made before and after Amicon column filtration were used to calculate [¹²⁵I]-IgG specific activity and average filtration recovery (approximately 80%); unlabeled ChromPure rat IgG was added to the ~1–2 mg/ml [¹²⁵I]-IgG solution to achieve the final dosing concentration, if needed. The final radiolabeled antibody ([¹²⁵I]-IgG) was found to be typically > 99% pure using Trichloroacetic acid precipitation.

2.3.2. Fluorescently labeled antibodies

Lyophilized non-targeted Alexa Fluor^® 488 labeled ChromPure rat IgG (AF488-IgG; Jackson Immunoresearch; catalog number: 012–540-003) was reconstituted in distilled water to obtain a 2 mg/ml solution. This AF488-IgG solution was concentrated using a 0.5 ml Amicon Ultra 100 kDa molecular weight cut-off centrifugal filter (Millipore Sigma). 0.5 ml of the 2 mg/ml AF488-IgG was added to the filter and centrifuged at 14,000 ×g for 30 min at 4 °C using an Eppendorf 5430R centrifuge with a FA-45–24-11-HS rotor. The filtrate was discarded and a recovery spin was performed at 1000 ×g for 2 min at 4 °C to obtain a ~30 mg/ml solution with ~70% filtration recovery. The concentrated AF488-IgG solution was used the same day and was administered intranasally.

Matrix metalloproteinase-9 (MMP-9):

activated rat MMP-9 (Sino Biological; catalog number: 80049-R08H) was diluted in saline and administered intranasally, as described previously [25].

2.4. Intranasal and intra-arterial administration paradigm

2.4.1. Intranasal dosing

Intranasal administration was performed similar to past reports [23], with minor modifications. Animals were laid in a supine position and non-invasively administered 48 μl of the antibody solution reconstituted in saline (administered as 4 drops of 12 μl each to alternate nares over 15 min, with a gap of 5 min between each drop). For experiments examining intranasal dose-response, [¹²⁵I]-IgG doses of 50 μg, 1 mg, and 2.5 mg were utilized, corresponding to [¹²⁵I]-IgG solution concentrations of 1 mg/ml, 20 mg/ml, and 50 mg/ml, respectively. To test the application of matrix metalloproteinase-9 (MMP-9) as a nasal absorption enhancer the following dosing paradigm was used: active MMP-9 (100 nM) solution reconstituted in saline was first administered intranasally as 4 drops of 6 μl each to alternate nares over 15 min, with a gap of 5 min between each drop, followed by administration of the antibody solution as 4 drops of 6 μl each in a similar manner. For MMP-9 pre-treatment experiments, the intranasal [¹²⁵I]-IgG dose was 50 μg and to ensure that this dose could be administered in 24 μl (i.e., half the previous volume) the concentration of [¹²⁵I]-IgG was doubled (2 mg/ml). Saline pre-treatment control experiments were performed in a separate set of animals to compare against the MMP-9 pre-treatment data. For fluorescence imaging experiments, the intranasal AF488-IgG dose was 0.7 mg and concentration of the AF488-IgG solution in saline was ~30 mg/ml. All intranasal AF488-IgG studies were performed with intranasal MMP-9 pre-administration.

2.4.2. Intra-arterial dosing

[¹²⁵I]-IgG solution was administered intra-arterially in saline as a 500 μl bolus dose over 1–2 min through the cannula inserted in the abdominal aorta, followed by a saline chaser injection of 500 μl to ensure that all of the antibody was washed out of the cannula dead volume and entered the systemic circulation. Intra-arterial [¹²⁵I]-IgG doses were selected to match the endpoint blood concentrations observed after intranasal dosing. These endpoint blood matched intra-arterial [¹²⁵I]-IgG doses were 0.13 μg, 2.6 μg, and 6.5 μg, respectively (see results for further explanation).

2.4.3. Euthanasia and perfusion-fixation of tissue

At 30 min after either intranasal or intra-arterial administration of [¹²⁵I]-IgG, animals used for tissue microdissection were euthanized by exsanguination via perfusion with 0.01 M phosphate buffered saline (PBS), pH 7.4 followed by perfusion with 2% paraformaldehyde (Alfa Aesar) and 2.5% glutaraldehyde (Thermo Fisher) in 0.1 M phosphate buffer (PB), pH 7.4. Animals used for CSF sampling were euthanized following CSF withdrawal with a 1 ml intracardial injection of 1 M KCl at 30 min following intranasal or intra-arterial [¹²⁵I]-IgG administration. At 30 min following intranasal AF488-IgG administration, animals were euthanized by exsanguination via perfusion with 0.01 M PBS, pH 7.4 followed by perfusion with 4% paraformaldehyde (Alfa Aesar) in 0.1 M PB, pH 7.4.

2.5. Methods to measure tissue [¹²⁵I]-IgG antibody concentration and distribution in the blood, CSF, and CNS

2.5.1. Blood sampling

Blood was sampled at ten minute intervals following intranasal or intra-arterial administration of [¹²⁵I]-IgG solution through the cannula inserted in the abdominal aorta.

2.5.2. CSF sampling via intracisternal withdrawal

Following the surgical insertion of cannulas into the cisterna magna and the abdominal aorta, [¹²⁵I]-IgG solution was administered intranasally or intra-arterially as described above. CSF was withdrawn at a constant flow rate of 5 μl/min, similar to the physiological rate of CSF production in rats [11]. Three separate CSF fractions (50 μl each) were sampled from 0–10, 10–20, to 20–30 min each following the administration of the first intranasal drop of antibody solution. CSF samples were spun in a centrifuge (Eppendorf 5430R) at 12,000 ×g for 5 min at 4 °C. 40 μl of supernatant was collected for measurement of antibody concentration in CSF. Lack of blood contamination was confirmed via both a visual absence of a red blood cell pellet following centrifugation as well as testing utilizing a Hemoglobin ELISA assay (Kamiya Biomedical, KT-460).

2.5.3. Microdissection of major ventral cerebral arteries

Following perfusion and fixation, the brain was extracted from the skull. The cerebral vasculature on the ventral brain surface was marked using India ink for better visibility. Cuts were made using Vannas scissors (Harvard Apparatus) at the left and right posterior communicating arteries under a stereo microscope (Olympus SZ61) (Fig. 1C). Anterior and posterior cerebral vasculature was peeled off the brain surface using blunt forceps and collected separately. The anterior cerebral vasculature consisted primarily of the middle and anterior cerebral arteries. The posterior cerebral vasculature consisted primarily of the basilar and vertebral arteries.

2.5.4. Brain and cervical spinal cord microdissection

As shown in Fig. 1A, 1 mm serial brain sections were made with a coronal precision rat brain matrix (Brain Tree Scientific Inc.). Typically, 5 sections anterior to bregma and 3 sections posterior to bregma were collected (Fig. 1 A1–A9). The coronal section at bregma was distinguished by the anterior commissure joined across the two hemi-spheres (Fig. 1 A6). The olfactory bulbs, midbrain, pons, medulla, cerebellum, and cervical spinal cord were collected as gross regions based on well-defined anatomical landmarks (Fig. 1A) [37, 38]. The cervical segment of the spinal cord was separated from the brainstem by making a cut along the spinal flexure using a scalpel (# 10 blade – Royal Tek) (Fig. 1A). Approximately 1 mm of the caudal end of the cervical spinal cord was cut and discarded to minimize possible contamination from the trachea that might occur during decapitation. The frontal cortex (Fig. 1 A1 and A2), motor cortex (Fig. 1 A2–A9), primary somatosensory cortex (Fig. 1 A3–A9), caudoputamen (Fig. 1 A4–A6), and hippocampus (Fig. 1 A8 and A9) were microdissected from the 1 mm serial coronal brain sections with a scalpel (# 10 blade – Royal Tek) or 1–2 mm sample corers (Fine Science Tools) using a micropunch technique [39]. Brain microdissections were made using the [40] Rat Brain Atlas as a guide. All brain dissections were performed with tissue visualized under a stereo microscope (Olympus SZ61).

2.5.5. Trigeminal nerve dissection

Trigeminal nerve segments were extracted from the skull base following brain removal. The ophthalmic (V1) and maxillary (V2) segments of the trigeminal nerve, extending from the anterior lacerated foramen anteriorly to the point where the nerve root contacts the brainstem posteriorly, were carefully dissected away from the mandibular segment (V3) (Fig. 1B). V1 and V2, but not V3, innervate the nasal mucosa [19, 23]. Dura was carefully peeled of the dissected trigeminal nerve segments using forceps.

2.5.6. Radioactivity measurement

Solid and fluid tissue samples were placed in a 5 ml tube and the wet weight was determined with a microbalance (Mettler Toledo, XP26) prior to gamma counting. Gamma counting was performed using a Packard Cobra II auto Gamma counter (Perkin Elmer). The specific activity of the [¹²⁵I]-IgG solution, tissue weight, and radioactivity counts per minute (CPM) values were used to determine the concentration of [¹²⁵I]-IgG in each tissue sample. For autoradiography, 300 μm thick coronal or sagittal brain sections were exposed to a storage phosphor screen (GE Healthcare, Life Sciences) in a light protected exposure cassette (GE Healthcare, Life sciences) for 7 weeks. The storage phosphor screen was scanned on a Typhoon phosphor-imager (GE Healthcare, Life sciences) at 50 μm resolution.

2.6. Investigating the intactness of intranasally administered [¹²⁵I]-IgG in the brain

Anesthetized rats were administered 50 μg [¹²⁵I]-IgG solution as described previously and subsequently euthanized at 30 min by exsanguination and perfusion with 0.01 M phosphate buffered saline (PBS), pH 7.4 to eliminate blood-borne signal. The brain soluble protein fraction was then used to analyze the intactness of radiolabeled IgG using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins by molecular weight followed by auto-radiography to detect radiolabeled protein bands. Following intranasal [¹²⁵I]-IgG administration and perfusion with PBS, brains were rapidly extracted and homogenized in lysis buffer (50 mM Tris, 5 mM EDTA, pH 8.00; 2 ml lysis buffer/g of brain) using a Dounce homogenizer on ice. A protease inhibitor cocktail tablet (Roche) was added to the lysis buffer to minimize protease activity. The brain homogenate was transferred to microcentrifuge tubes and subjected to centrifugation at 14,000 ×g at 4 °C for 1h using an Eppendorf 5430R centrifuge with a FA-45–24-11-HS rotor. Brain homogenate supernatant, i.e., the brain soluble protein fraction, was collected in a separate tube, mixed with (4×) NuPAGE loading dye and heated at 70 °C for 10 min in a water bath to denature the protein. Denatured brain soluble protein fractions were then subjected to non-reducing SDS-PAGE to separate proteins by molecular weight. Radioactivity counts per minute (CPM) measurements of the brain soluble protein fraction were used to match the amount of radiolabeled protein loaded into each well. This was also done to be able to roughly estimate the amount of [¹²⁵I]-IgG to be loaded in a separate well as a positive control. For SDS-PAGE, protein samples were run on NuPAGE 4–12% Bis-Tris gel (1.5 mm × 10 wells), with a NuPAGE 3-(N-morpholino)-propanesulfonic acid (MOPS) running buffer (50 mM MOPS, 50 mMTris base, 0.1% SDS, 1 mM EDTA, pH 7.7), at a constant voltage of 200 V and approximate current ranging from 110 to 125 mA (at run start) to 70–80 mA (at run end) for 35–50 min. Following SDS-PAGE, the polyacrylamide gel was rinsed three times with deionized water and then incubated in a gel drying solution (20% ethanol and 5% glycerol in deionized water) for 30 min. Cellophane sheets were wetted first in deionized water for 1–2 min and next in gel drying solution for about 30 s. The DryEase^® Gel Drying Frame was used to dry the polyacrylamide gel sandwiched between a top and bottom layer of pre-wetted cellophane for 24 h at room temperature. The dried polyacrylamide gel was retained between the top and bottom layers of cellophane and removed from the geldrying frame. Excess cellophane was trimmed away. For autoradiography to detect the radiolabeled protein bands, the polyacrylamide gel was exposed to a storage phosphor screen (GE Healthcare, Life Sciences) in a light protected exposure cassette (GE Healthcare, Life sciences) for 3 weeks. The storage phosphor screen was scanned on a Typhoon phosphor-imager (GE Healthcare, Life sciences) at a 200 μm resolution with a setting selected for best sensitivity.

2.7. Fluorescence imaging to study AF488-IgG distribution in the nasal passages, trigeminal nerves, and brain

2.7.1. Ex vivo fluorescence microscopy

Following perfusion fixation, the lateral walls of the nasal passages and the skull base were imaged via ex vivo imaging performed on an Olympus MVX10 Macroview microscope equipped with an Orca-flash 2.8 CMOS camera (Hamamatsu) and Lumen Dynamics X-Cite 120Q illuminator using the appropriate filter set (Chroma, U-M49002XL).

2.7.2. Immunofluorescence and confocal microscopy

Following perfusion fixation, brains were extracted and post-fixed overnight in 4% paraformaldehyde (in 0.1 M PB, pH 7.4) at 4 °C. Brains were cut into 100 μm coronal sections using a vibratome (Leica VT100S, Wetzlar, Germany) and free-floating sections were stored in 0.01 M phosphate buffered saline (PBS), pH 7.4 at 4 °C. Nasal passages and trigeminal nerves were cryoprotected in 20% sucrose (in PBS, pH 7.4) for 24h at 4°C, embedded in optimal cutting temperature (OCT – Tissue Tek) embedding medium, and then snap frozen in absolute isopentane (Alfa Aesar) chilled on dry ice. Frozen tissue was then cut into 25 μm coronal sections using a cryostat (Leica CM 1950) and mounted on glass slides (Thermo Fisher). Frozen tissue sections mounted on glass slides was stored at −20 °C.

Immunofluorescence was performed on free-floating brain sections and on nasal mucosa and trigeminal nerve tissue cryosections mounted on glass slides. All tissue sections were washed in PBS (three washes for 5 min each), pH 7.4 and blocked for 1 h in 3% serum of species that secondary antibody was raised in (goat or donkey serum - Sigma) in PBS with 0.1% Triton X-100, pH 7.4 (blocking buffer). 5% Bovine Serum Albumin (Sigma Aldrich) in PBS with 0.5% Triton X-100 was used as a blocking buffer when staining tissue with antibody against alpha smooth muscle actin (ɑ-SMA). Tissue sections were then incubated in blocking buffer with appropriate primary antibodies over-night at 4 °C. Next, tissue sections were washed in PBS with 0.1% Triton X-100 (PBST) three times for 5 min each and then incubated in blocking buffer with secondary antibodies at room temperature for 2 h. This was followed by 3 washes in PBST. In some instances sections were then incubated with 4′,6-diamidino-2-phenylindole (DAPI) in PBS (2 μg/ml) for 20 min to label cell nuclei, followed by 3 more washes in PBS. After a final wash in PBS, free-floating brain sections were mounted onto glass slides. All tissue section slides were coverslipped in ProLong Diamond (Thermo Fisher) mounting medium. Primary antibodies used included the following: mouse anti-rat endothelial cell antigen-1 (RECA-1) monoclonal antibody (Abcam ab9774; 1:500 dilution), rabbit anti-protein gene product 9.5 (PGP 9.5) polyclonal antibody (Abcam ab27053; 1:1000), chicken anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (Novus NBP1–05198; 1:1000), and Cy3 labeled mouse anti-alpha smooth muscle actin (ɑ-SMA) monoclonal antibody (Sigma Aldrich C6198; 1:500). Secondary antibodies used included the following: goat anti-mouse pre-adsorbed AlexaFluor 647 (Abcam ab150119; 1:500), goat anti-rabbit AlexaFluor 594 (Life Technologies ab150080; 1:500), and goat anti-chicken AlexaFluor 594 (Abcam ab150172; 1:500). Laser scanning confocal microscopy was performed using an Olympus FV1000 confocal microscope using FLUOVIEW software or a Nikon A1R confocal microscope with NIS Elements software. Images were optimized in Adobe Photoshop (Adobe Systems) with RGB histogram adjustments performed only on cellular marker channels to optimize signal. Signal in the channel corresponding to intranasally applied antibody fluorescence was left unaltered. No changes were made to the gamma values for any image. The purpose of fluorescence imaging was to delineate spatial distribution and localization of IgG. No quantification was performed using fluorescence images from this study.

3. Results

3.1. Intranasal and intra-arterial delivery of different doses of [¹²⁵I]-IgG resulted in similar endpoint blood concentrations

Intranasally applied macromolecules may access the CNS in one of two ways: (i) they may be absorbed into the systemic circulation via nasal blood vessels and access the CNS by crossing the BBB and/or BCSFBs; or (ii) they may access the CNS via direct perineural, perivascular, and lymphatic pathways that exist in the nasal mucosa [19, 23–25, 42]. Utilizing a strategy adapted from previous studies investigating intranasal delivery of other [¹²⁵I]-labeled and fluorescently labeled protein and dextran tracers to the CNS [23, 25], we first determined an intra-arterial dose that resulted in a similar endpoint (30 min) blood [¹²⁵I]-IgG concentration as that seen after a fixed intranasal dose of [¹²⁵I]-IgG in order to allow us to evaluate whether [¹²⁵I]-IgG detected in the CNS originated from the blood following intranasal delivery. We henceforth refer to such an intra-arterial dose as an ‘endpoint blood matched intra-arterial dose’. Animals receiving an endpoint blood matched intra-arterial dose of [¹²⁵I]-IgG therefore provided a measure of [¹²⁵I]-IgG penetration into the CNS and/or into the endothelial cell compartment of cerebral blood vessels originating from [¹²⁵I]-IgG in the bloodstream for both intra-arterial and intranasal dosing. Thus comparing the blood and CNS concentrations arising from an intranasal dose versus an endpoint blood matched intra-arterial dose in separate animals allowed us to delineate [¹²⁵I]-IgG targeting to the CNS as a result of access to direct nose-to-brain pathways arising within the nasal mucosa following intranasal delivery. We initially characterized the quantitative CNS distribution of [¹²⁵I]-IgG following a low tracer intranasal dose (50 μg) and compared this to the quantitative CNS distribution following an endpoint blood matched intra-arterial dose (0.13 μg). Table 1 indicates that the endpoint blood concentrations following this low tracer intranasal dose and endpoint blood matched intra-arterial dose were not significantly different (n.s.), as intended (intranasal to intra-arterial endpoint blood [¹²⁵I]-IgG concentration ratio = 1.1, t(9) = 0.430, p = .677 (n.s.), two tailed Studenťs t-test); furthermore, the area under the blood concentration-time curve (AUC) over the 30 min duration of the experiment (Table 1) was 2.6-fold higher following the endpoint blood matched intra-arterial dose versus the low tracer intranasal dose (t(8) = −8.098, p = .001, two tailed Studenťs t-test), suggesting total blood exposure for the intra-arterial dose actually exceeded that with the tracer intranasal dose. Similar intranasal and matched intra-arterial dose pairs were also delineated to test the effects of two higher intranasal and intra-arterial doses on CNS [¹²⁵I]-IgG penetration. Table 2 indicates that the endpoint blood concentrations following a 1 mg intranasal dose (20-fold higher than intranasal tracer) and the corresponding endpoint blood matched intra-arterial dose (2.6 μg) were not significantly different, as intended (intranasal to intra-arterial endpoint blood [¹²⁵I]-IgG concentration ratio = 1.3, t(10) = 1.099, p = .298 (n.s.), two tailed Student’s t-test); the AUC over the 30 min duration of the experiment (Table 2) was 2.1-fold higher following the endpoint blood matched intra-arterial dose versus the 1 mg intranasal dose (U = 2, p = .009, Mann-Whitney rank sum test). Likewise, the endpoint blood concentrations following a 2.5 mg intranasal dose (50-fold higher than intranasal tracer) and the corresponding endpoint blood matched intra-arterial dose (6.5 μg) (Table 3) were not significantly different, as intended (intranasal to intra-arterial endpoint blood [¹²⁵I]-IgG concentration ratio = 1, t (8) = 0.219, p = .832 (n.s.), two tailed Student’s t-test); the AUC over the 30 min duration of the experiment (Table 3) was 3-fold higher following the endpoint blood matched intra-arterial dose versus the 2.5 mg intranasal dose (t(8) = −7.531, p = .001, two tailed Student’s t-test). Taken together, the data indicate similar endpoint blood concentrations were obtained for the three intranasal/intra-arterial dose pairs, with greater overall blood exposure for the intra-arterial dosing compared to intranasal dosing at each dose level. These intranasal/intra-arterial dose pairs facilitated comparison of differences in CNS penetration following intranasal dosing versus the endpoint blood matched intra-arterial dosing, allowing higher levels obtained following intranasal delivery to be interpreted as delivery of [¹²⁵I]-IgG to the CNS via pathways unique to the intranasal route.

Table 1.

Tissue concentrations^a (pM) of [¹²⁵I]-IgG following intranasal or intra-arterial administration (low tracer dose).

	Intranasal dose (50 μg)	Intra-arterial dose (0.13 μg)
Blood	(pM)	(pM)
0 min	0 (6)	80.22 ± 1.55 (7)
10 min	13.69 ± 1.65 (6)	64.72 ± 7.44 (7)
20 min	34.29 ± 4.43 (5)	62.69 ± 7.03 (7)
30 min	57.35 ± 8.29 (6)	52.89 ± 5.33 (7)
Blood AUC (0–30 min)	(pM*min)	(pM*min)
	732.17 ± 97.73 (5)^d	1877.38 ± 102.21(5)
Cerebrospinal fluid (CSF)	(pM)	(pM)
0–10 min	0.06 ± 0.01 (3) ^Γ	0.05 ± 0.04 (3)
10–20 min	0.55 ± 0.07 (3) ^e^Γ	0.22 ± 0.04 (3)
20–30 min	0.89 ± 0.19 (3) ^f^Γ	0.25 ± 0.11 (3)
Nervous system regions	(pM)	(pM)
ACA & MCA ^b	30.02 ± 9.31 (6) ^g	2.52 ± 1.21 (7)
Basilar & vertebral arteries ^c	11.43 ± 4.74 (5)	4.72 ± 3.79 (7)
Olfactory bulbs	34.25 ± 6.96 (6) ^h	0.17 ± 0.07 (7)
Frontal cortex	6.4 ± 2.00 (6) ^h	0.39 ± 0.18 (7)
Caudoputamen	1.28 ± 0.37 (6)	0.55 ± 0.47(6)
Motor cortex	2.51 ± 1.08 (6)	0.77 ± 0.60 (7)
Primary somatosensory cortex	2.25 ± 0.99 (5)	0.41 ± 0.21 (7)
Midbrain	1.67 ± 0.28 (6) ⁱ	0.70 ± 0.50 (7)
Hippocampus	2.02 ± 0.38 (6) ^j	0.26 ± 0.11 (7)
Medulla	1.71 ± 0.27 (6) ^k	0.13 ± 0.11 (7)
Pons	2.07 ± 0.43 (6) ^h	0.10 ± 0.04 (7)
Cerebellum	1.72 ± 0.24 (6) ^h	0.16 ± 0.11 (7)
Cervical spinal cord	2.26 ± 0.79 (6) ^l	0.14 ± 0.09 (7)
Trigeminal nerves	32.34 ± 9.01 (5) ^m	0.38 ± 0.25 (5)
Lymph nodes	(pM)	(pM)
Deep cervical lymph nodes	103.02 ± 56.56 (4) ⁿ	0.64 ± 0.23 (7)
Superficial cervical lymph nodes	68.28 ± 31.82 (5) ^o	0.86 ± 0.20 (7)
Axillary lymph nodes	4.60 ± 1.43 (4) ⁿ	0.68 ± 0.12 (7)

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Blood, cerebrospinal fluid (CSF), nervous system, and lymph node radiolabel concentrations following intranasal or intra-arterial administration of [¹²⁵I]-IgG (low tracer doses). Intranasal administration of a low tracer dose (50 μg) was performed drop wise (4 drops of 12 μl each) over approximately 15 min. Intra-arterial administration of a dose (0.13 μg) that resulted in similar end point blood concentrations as the intranasal dose was performed as a bolus dose (500 μl) over 1–2 min. The concentration at time zero following intra-arterial administration of [¹²⁵I]-IgG was estimated by extrapolation. Blood and CSF were sampled over 30 min following [¹²⁵I]-IgG administration while brain tissue was sampled at 30 min following [¹²⁵I]-IgG administration.

Data are presented as mean ± S.E.M. (n independent experiments);

anterior cerebral and middle cerebral arteries – isolated perfused vessels (indirect measure of perivascular compartment);

isolated perfused posterior circulation vessels mainly comprising the basilar and vertebral arteries (a small portion of the proximal posterior cerebral and superior cerebellar arteries are also included; indirect measure of perivascular compartment); Statistical analysis for tissue samples - intranasal vs intra-arterial dosing: Two-tailed Studenťs t-test –

t (8) = −8.098, p = .001;

t(4) = −4.168, p = .014;

t(4) = −2.971, p = .041;

t(11) = 3.177, p = .009;

Mann-Whitney U = 0, p = .001;

ⁱ

Mann-Whitney U = 6, p = .035;

t(11) = 4.767, p = .001;

t(11) = 5.755, p = .001; Mann-Whitney Rank sum test –

Mann-Whitney U = 1, p = .002;

Mann-Whitney U = 0, p = .008;

ⁿ

Mann-Whitney U = 0, p = .006;

Mann-Whitney U = 0, p = .003.

^Γ

One – way ANOVA across 3 CSF fractions collected over 30 min following intranasal delivery: F(2, 6) = 13.39, p = .006, post hoc analysis: Bonferroni t-test, p = .006, difference between 0 and 10 min and 20–30 min fractions. No difference observed between CSF fractions following intra-arterial delivery.

Table 2.

Tissue concentrations^a (pM) of [¹²⁵I]-IgG following intranasal or intra-arterial administration (middle dose).

	Intranasal dose (1 mg)	Intra-arterial dose (2.6 μg)
Blood	(pM)	(pM)
0 min	0 (6)	1465.05 ± 87.27 (6)
10 min	239.61 ± 55.69 (6)	1086.02 ± 94.06 (6)
20 min	724.60 ± 171.93 (6)	975.01 ± 89.29 (6)
30 min	1150.97 ± 194.40 (6)	917.77 ± 85.04 (6)
Blood AUC (0–30 min)	(pM*min)	(pM*min)
	15,396.90 ± 3216.89 (6)	32,524.37 ± 2497.53 (6)
Nervous system regions	(pM)	(pM)
ACA & MCA ^b	1280 ± 461.5 (6) ^d	4.02 ± 1.03 (6)
Basilar & vertebral arteries ^c	1177.49 ± 382.34 (5) ^e	2.77 ± 1.26 (6)
Olfactory bulbs	720.77 ± 246.14 (6) ^d	0.53 ± 0.10 (6)
Frontal cortex	154.62 ± 47.67 (6) ^d	0.34 ± 0.10 (6)
Caudoputamen	133.82 ± 58.28 (6) ^d	1.49 ± 0.71(6)
Motor cortex	135.76 ± 50.03 (6) ^d	0.47 ± 0.07 (6)
Primary somatosensory cortex	209.42 ± 21.47 (5) ^e	0.54 ± 0.18 (6)
Midbrain	38.53 ± 9.38 (6) ^d	0.34 ± 0.50 (6)
Posterior hippocampus	45.44 ± 8.14 (6) ^d	0.33 ± 0.06 (6)
Medulla	107.42 ± 30.58 (6) ^d	0.26 ± 0.06 (6)
Pons	55.60 ± 14.99 (6) ^d	0.34 ± 0.06 (6)
Cerebellum	40.39 ± 9.80 (6) ^d	0.56 ± 0.20 (6)
Cervical spinal cord	334.35 ± 224.67 (6) ^d	0.25 ± 0.13 (6)
Trigeminal nerves	1706.60 ± 779.57 (4) ^f	3.66 ± 1.13 (6)
Lymph nodes	(pM)	(pM)
Deep cervical lymph nodes	3020.64 ± 1432.84 (5) ^e	16.02 ± 5.05 (6)
Superficial cervical lymph nodes	2108.56 ± 934.36 (6) ^d	9.86 ± 0.58 (6)
Axillary lymph nodes	202.98 ± 42.48 (6) ^d	8.87 ± 0.57 (6)

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Blood, nervous system, and lymph node radiolabel concentrations following intranasal or intra-arterial administration of [¹²⁵I]-IgG (middle doses). Intranasal administration of a middle (20-fold higher than tracer) dose (1 mg) was performed drop wise (4 drops of 12 μl each) over approximately 15 min. Intra-arterial administration of a dose (2.6 μg) that resulted in similar end point blood concentrations as the intranasal dose was performed as a bolus dose (500 μl) over 1–2 min. The concentration at time zero following intra-arterial administration of [¹²⁵I]-IgG was estimated by extrapolation.

Data are presented as mean ± S.E.M. (n independent experiments);

anterior cerebral and middle cerebral arteries – isolated perfused vessels (indirect measure of perivascular compartment);

Mann-Whitney U = 0, p = .002;

Mann-Whitney U = 0, p = .004;

Mann-Whitney U = 0, p = .010.

Table 3.

Tissue concentrations^a (pM) of [¹²⁵I]-IgG following intranasal or intra-arterial administration (high dose).

	Intranasal dose (2.5 mg)	Intra-arterial dose (6.5 μg)
Blood	(pM)	(pM)
0 min	0 (4)	4324.09 ± 73.67 (6)
10 min	486.91 ± 101.71 (4)	3691.64 ± 280.2 (6)
20 min	1484.94 ± 323.69 (4)	3322.45 ± 212.17 (6)
30 min	2663.77 ± 357.96 (4)	2588.16 ± 158.35 (6)
Blood AUC (0–30 min)	(pM*min)	(pM*min)
	33,037.40 ± 5948.52 (4)	99,209.03 ± 5937.43 (6)
Nervous system regions	(pM)	(pM)
ACA & MCA ^b	1510.13 ± 767.27 (5) ^d	4.15 ± 1.49 (6)
Basilar & vertebral arteries ^c	1581.38 ± 511.51 (5) ^d	7.59 ± 3.20 (6)
Olfactory bulbs	4418.54 ± 1862.50 (6) ^e	2.53 ± 0.61 (6)
Frontal cortex	547.44 ± 100.56 (6) ^e	2.04 ± 0.33 (6)
Caudoputamen	277.31 ± 63.95 (6) ^e	2.39 ± 0.71 (6)
Motor cortex	275.13 ± 48.4 (6) ^e	1.62 ± 0.43 (6)
Primary somatosensory cortex	226.06 ± 41.24 (5) ^e	2.40 ± 0.41 (6)
Midbrain	330.87 ± 69.00 (6) ^e	1.70 ± 0.30 (6)
Posterior hippocampus	276.28 ± 53.39 (6) ^e	1.78 ± 0.41 (6)
Medulla	424.54 ± 99.62 (6) ^e	1.84 ± 0.43 (6)
Pons	346.00 ± 51.44 (6) ^e	2.11 ± 0.44(6)
Cerebellum	249.73 ± 52.55 (6) ^e	1.77 ± 0.37 (6)
Cervical spinal cord	384.47 ± 68.31 (5) ^d	2.43 ± 1.18(6)
Trigeminal nerves	15,544.35 ± 5506.92 (6) ^e	20.71 ± 3.81 (6)
Lymph nodes	(pM)	(pM)
Deep cervical lymph nodes	1109.34 ± 341.06 (6) ^f	255.99 ± 118.91 (6)
Superficial cervical lymph nodes	53.97 ± 14.46 (6)	2720.47 ± 353.65 (7) ^g
Axillary lymph nodes	896.07 ± 399.82 (6) ^g	38.89 ± 11.18 (6)

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Blood, nervous system, and lymph node radiolabel concentrations following intranasal or intra-arterial administration of [¹²⁵I]-IgG (high doses). Intranasal administration of a high (50-fold higher than tracer) dose (2.5 mg) was performed drop wise (4 drops of 12 μl each) over approximately 15 min. Intra-arterial administration of a dose (6.5 μg) that resulted in similar end point blood concentrations as the intranasal dose was performed as a bolus dose (500 μl) over 1–2 min. The concentration at time zero following intra-arterial administration of [¹²⁵I]-IgG was estimated by extrapolation.

Data are presented as mean ± S.E.M. (n independent experiments);

anterior cerebral and middle cerebral arteries – isolated perfused vessels (indirect measure of perivascular compartment);

Mann-Whitney U = 0, p = .004;

Mann-Whitney U = 0, p = .002;

t(10) = 2.363, p = .04. Mann-Whitney Rank sum test –

Mann-Whitney U = 0, p = .001.

3.2. A low 50 μg tracer [¹²⁵I]-IgG dose accessed the CNS and bypassed the BBB and BCSFBs following intranasal administration

Gamma counting was used to determine [¹²⁵I]-IgG concentrations in the CSF (withdrawn from anesthetized animals) over time during and following the administration of the low tracer intranasal dose and corresponding endpoint blood matched intra-arterial dose. For this same low tracer intranasal dose and endpoint matched intra-arterial dose, gamma counting was performed on a large number of nervous system tissue samples (collected post-exsanguination by perfusion with saline and aldehyde fixation) at 30 min post-administration in separate animals. The [¹²⁵I]-IgG concentrations reaching most nervous system regions and the CSF were significantly higher following the administration of the low tracer intranasal dose compared to the endpoint blood matched intra-arterial dose (Table 1). For example, the concentration of [¹²⁵I]-IgG was ~200-fold higher in the olfactory bulbs (U = 0, p = .001, Mann-Whitney rank sum test) and ~85-fold higher in the trigeminal nerves (U = 0; p = .008, Mann-Whitney rank sum test) following the low tracer intranasal dose versus the endpoint blood matched intra-arterial dose (Table 1). This suggests an intranasal route specific [¹²⁵I]-IgG access to olfactory and trigeminal nerve associated pathways that bypassed the BBB and BCSFBs to target the CNS, similar to what has been described for smaller macromolecules delivered intranasally in rodents [23, 25] and non-human primates [24]. [¹²⁵I]-IgG concentrations associated with isolated major cerebral blood vessels (saline and fixative perfused) provided a measure of cellular uptake by endothelial cells from the bloodstream as well as any [¹²⁵I]-IgG that may have accessed the perivascular space compartment associated with the tunica media and adventitia [34, 43]. We measured ~12-fold higher [¹²⁵I]-IgG concentrations in dissected samples containing both the anterior cerebral artery (ACA) and middle cerebral artery (MCA) following the low tracer intranasal dose versus the endpoint blood matched intra-arterial dose (t(11) = 3.177, p = .009, two tailed Studenťs t-test; Table 1); measured levels in isolated vessels from the posterior circulation (basilar and vertebral arteries) trended higher following intranasal delivery as well although the differences were not significant. These results are consistent with intranasal but not intra-arterial [¹²⁵I]-IgG gaining significant entry into the perivascular compartment, providing a potential route for widespread intranasal [¹²⁵I]-IgG distribution within the brain, similar to that observed previously for intranasally delivered dextrans [25] and other routes of central IgG delivery such as intrathecal administration [34]. Significantly higher [¹²⁵I]-IgG concentrations were also detected in several CNS regions such as the midbrain (U = 6, p = .035, Mann-Whitney rank sum test), hippocampus (t(11) = 4.767, p = .001, two tailed Studenťs t-test), and cervical spinal cord (U = 1, p = .002, Mann-Whitney rank sum test) following the low tracer intranasal dose versus the endpoint blood matched intra-arterial dose (Table 1). Furthermore, intranasal dosing yielded an apparent gradient in [¹²⁵I]-IgG levels decreasing in the rostral-to-caudal direction along the neural axis, whereas endpoint blood matched intra-arterial [¹²⁵I]-IgG dosing yielded uniform, low levels from the olfactory bulbs to the spinal cord.

Data in Table 1 demonstrates that the concentration of [¹²⁵I]-IgG increased significantly over 30 min in the CSF following the administration of the low tracer intranasal dose in separate experiments (F(2, 6) = 13.39, p = .006, One-way ANOVA, post hoc analysis: Bonferroni t-test, p = .006, significant difference between 0 and 10 and 20–30 min fractions); such an increase did not occur following the endpoint blood matched intra-arterial dose (p = .180 (n.s.), One-way ANOVA). The [¹²⁵I]-IgG concentration at 30 min in the CSF was ~8-fold higher following low tracer intranasal versus the endpoint blood matched intra-arterial dose (t(4) = −2.971, p = .041, two tailed Studenťs t-test) (Table 1). The endpoint CSF: blood ratio following intranasal delivery (~0.016) was nearly 3-fold higher than that following intra-arterial delivery (~0.005).

3.3. Two higher [¹²⁵I]-IgG doses (1 mg and 2.5 mg) accessed the CNS and bypassed the BBB and BCSFBs following intranasal administration

In order to investigate the relationship between [¹²⁵I]-IgG dose and the corresponding change (i.e., response) in [¹²⁵I]-IgG concentrations in the brain following intranasal versus endpoint blood matched intra-arterial doses, we intranasally administered 1 mg [¹²⁵I]-IgG (20-fold higher than intranasal tracer) and 2.5 mg [¹²⁵I]-IgG (50-fold higher than intranasal tracer) doses along with corresponding endpoint blood matched intra-arterial doses in four different sets of animals, as described above. We observed that at 30 min following the 1 mg and 2.5 mg intranasal doses, the concentrations of [¹²⁵I]-IgG reaching most brain regions were significantly greater than that following the corresponding endpoint blood matched intra-arterial doses (Tables 2 and 3). For example, the concentration of [¹²⁵I]-IgG was ~1370-fold higher in the olfactory bulbs (U = 0, p = .002, Mann-Whitney rank sum test) and ~470-fold higher in the trigeminal nerves (U = 0, p = .010, Mann-Whitney rank sum test) following the 1 mg intranasal dose and ~1750-fold higher in the olfactory bulbs (U = 0, p = .002, Mann-Whitney rank sum test) and ~750-fold higher in the trigeminal nerves (U = 0, p = .002, Mann-Whitney rank sum test) following the 2.5 mg intranasal dose than that following the corresponding endpoint blood matched intra-arterial doses (Tables 2 and 3). This result is consistent with an intranasal route specific [¹²⁵I]-IgG access to olfactory and trigeminal nerve associated pathways that bypass the BBB and BCSFBs to enter the CNS. [¹²⁵I]-IgG concentrations associated with isolated major cerebral blood vessels provided an indirect measure of [¹²⁵I]-IgG cellular uptake by endothelial cells and levels in the perivascular compartment. [¹²⁵I]-IgG concentrations associated with the anterior and middle cerebral arteries were ~320-fold higher following the 1 mg (U = 0, p = .002, Mann-Whitney rank sum test) and ~360-fold higher following the 2.5 mg (U = 0, p = .004, Mann-Whitney rank sum test) intranasal doses compared to the corresponding endpoint blood matched intra-arterial doses (Tables 2 and 3). Similarly, [¹²⁵I]-IgG concentrations associated with the basilar and vertebral arteries were ~425-fold higher following the 1 mg (U = 0, p = .004, Mann-Whitney rank sum test) and ~210-fold higher following the 2.5 mg (U = 0, p = .004, Mann-Whitney rank sum test) intranasal doses compared to the corresponding endpoint blood matched intra-arterial doses (Tables 2 and 3). This result is consistent with significantly greater [¹²⁵I]-IgG access to the perivascular spaces associated with cerebral blood vessels following the 1 and 2.5 mg intranasal doses versus the endpoint blood matched intra-arterial doses. Significantly higher concentrations of [¹²⁵I]-IgG were also detected in several therapeutically relevant brain regions such as the midbrain^a,b, hippocampus^a,b, and cervical spinal cord^a,c following the 1 mg (^aU = 0, p = .002, Mann-Whitney rank sum test) and 2.5 mg (^bU = 0, p = .002; ^cU = 0, p = .004, Mann-Whitney rank sum test) intranasal doses versus the corresponding endpoint blood matched intra-arterial doses (Tables 2 and 3).

Overall, Tables 1–3 collectively provide evidence for significantly higher concentrations of [¹²⁵I]-IgG reaching several brain regions and the perivascular space associated with the major cerebral blood vessels following intranasal versus corresponding endpoint blood matched intra-arterial dosing at three different doses.

3.4. [¹²⁵I]-IgG heavy and light chains were detected in the brain soluble protein fraction following intranasal delivery

Supplementary Fig. 2 shows that [¹²⁵I] signal observed in the brain following intranasal administration of [¹²⁵I]-IgG could be attributed to [¹²⁵I]-labeled protein. SDS-PAGE and autoradiographic analysis of the brain soluble protein fraction yielded distinct heavy and light chain [¹²⁵I]-labeled protein bands following intranasal administration of [¹²⁵I]-IgG. Such detection of heavy and light chain bands has generally been considered consistent with intact IgG being present in vivo, e.g., in studies analyzing endogenous and exogenous IgG content in the brain [44].

3.5. Dose-response: intranasal but not intra-arterial administration of higher [¹²⁵I]-IgG doses resulted in significantly higher CNS IgG concentrations

In order to clarify the dose-response relationship following intranasal versus endpoint blood matched intra-arterial doses, it is instructive to ask how blood and CNS tissue [¹²⁵I]-IgG concentrations changed following higher doses with respect to the low tracer dose via both routes of delivery. For convenience, we will henceforth refer to the 50 μg (low tracer) intranasal dose as the ‘low intranasal dose’, the 1 mg intranasal dose as the ‘middle intranasal dose’, the 2.5 mg intranasal dose as the ‘high intranasal dose’, and the corresponding endpoint blood matched intra-arterial doses of 0.13 μg, 2.6 μg, and 6.5 μg as the ‘low intra-arterial dose’, ‘middle intra-arterial dose’, and ‘high intra-arterial dose’ respectively. Fig. 2 summarizes the fold change in blood and nervous system concentrations following the middle and high doses with respect to the corresponding low doses following intranasal and endpoint blood matched intra-arterial dosing. Endpoint blood [¹²⁵I]-IgG concentrations following the middle and high intranasal and intra-arterial doses were ~20-fold and 50-fold higher than the corresponding intranasal and intra-arterial low doses. This suggests that [¹²⁵I]-IgG concentrations in the blood compartment scaled proportionally with dose following both intranasal and intra-arterial delivery. In contrast, fold changes in [¹²⁵I]-IgG concentrations in sampled nervous system tissue exhibited much more complexity; in general, brain, cervical spinal cord, and large caliber cerebral blood vessel sample concentrations increased markedly following intranasal [¹²⁵I]-IgG but not intra-arterial [¹²⁵I]-IgG at higher doses. For example, the concentrations of [¹²⁵I]-IgG in the olfactory bulbs^a, midbrain^a, and hippocampus^a were ~20-fold higher in animals receiving the middle intranasal dose versus the low intranasal dose, i.e., [¹²⁵I]-IgG concentrations in these brain areas scaled proportionally with the 20-fold dose change (Fig. 2; ^aU = 0, p = .002, Mann-Whitney rank sum test). Other nervous system samples in animals receiving the middle intranasal dose such as the caudoputamen^a, cervical spinal cord^a, trigeminal nerves^b, anterior and middle cerebral arteries^a, and basilar and vertebral arteries^c (samples of leptomeningeal arteries and associated supporting tissue provided an indirect measure of [¹²⁵I]-IgG cellular uptake by endothelial cells and levels in the perivascular compartment) exhibited [¹²⁵I]-IgG concentrations that were ~40-fold to 150-fold higher than those with the low intranasal dose, i.e., [¹²⁵I]-IgG concentrations in these regions exceeded the 20-fold dose change (Fig. 2; ^aU = 0, p = .002; ^bU = 0, p = .016; ^cU = 0, p = .008; Mann-Whitney rank sum test). Conversely, the olfactory bulbs^a and hippocampus^b in animals receiving the middle intra-arterial dose exhibited [¹²⁵I]-IgG concentrations that were only ~1.2-fold to 3-fold higher than in the low intra-arterial dose group (Fig. 2; ^aU = 4, p = .014; ^bU = 0, p = .002, Mann-Whitney rank sum test) while other samples in the middle intra-arterial dose group failed to show a significant [¹²⁵I]-IgG concentration increase.

Fig. 2. — Dose-response: fold-change in [¹²⁵I]-IgG concentrations reaching the nervous system and blood following twenty and fifty-fold higher doses compared to the low tracer dose administered via the intranasal or intra-arterial route. Fold-change in [¹²⁵I]-IgG tissue concentrations derived from data presented as mean ± S.E.M in Tables 1 to 3. Intranasal: Low tracer dose: 50 μg; Middle dose (20-fold higher than tracer: 1 mg); High dose (50-fold higher than tracer: 2.5 mg). Intra-arterial: Low tracer dose: 0.13 μg; Middle dose (20-fold higher than tracer: 2.6 μg); High dose (50-fold higher than tracer: 6.5 μg). Grey lines indicate fold-change with respect to the low tracer intranasal and intra-arterial dose data. Abbreviations: pt. – point; ACA – anterior cerebral artery; MCA – middle cerebral artery; Bas. – Basilar artery; Vert. – Vertebral arteries; PVS – perivascular space; cerv. – cervical; IN – intranasal; IA – intra-arterial.

[¹²⁵I]-IgG concentrations in the anterior and middle cerebral arteries were ~50-fold higher in animals receiving the high intranasal dose versus the low intranasal dose – demonstrating that the fold change in [¹²⁵I]-IgG concentration in this region scaled proportionally with the 50-fold change in the intranasal dose (Fig. 2; U = 0, p = .004, Mann-Whitney rank sum test). [¹²⁵I]-IgG concentrations in other nervous system samples such as the olfactory bulbs^a, midbrain^a, hippocampus^a, caudoputamen^a, cervical spinal cord^b, trigeminal nerves^b, and basilar and vertebral arteries^c were ~100-fold to 500-fold higher in animals receiving the high intranasal dose versus the low intranasal dose, well exceeding the 50-fold dose change (Fig. 2; ^aU = 0, p = .002; ^bU = 0, p = .004; ^cU = 0, p = .008 Mann-Whitney rank sum test). Conversely, in animals receiving the high intra-arterial dose, nervous system tissue samples (with the exception of the trigeminal nerves) exhibited a minimal fold [¹²⁵I]-IgG concentration increase if any versus the low intra-arterial dose, well below the 50-fold dose change. The trigeminal nerves were an exception with [¹²⁵I]-IgG concentrations that were ~50-fold higher in animals receiving the high intra-arterial dose versus the low intra-arterial dose, in line with the 50-fold dose increase (Fig. 2; ^bU = 0, p = .004, Mann-Whitney rank sum test). Overall, the fold changes in concentrations of [¹²⁵I]-IgG detected in the nervous system, with the exception of the trigeminal nerves at the high intra-arterial dose, were lower than the fold changes in endpoint blood levels for the higher intra-arterial doses compared to the low tracer intra-arterial dose. Conversely, the fold changes in concentrations of [¹²⁵I]-IgG detected in the nervous system were either similar to or higher than the fold changes in endpoint blood levels for the higher intranasal doses compared to the low tracer intranasal dose, strongly suggesting intranasal [¹²⁵I]-IgG penetration into the CNS occurs via pathways to the CNS that are less saturable and distinct from those accessed following intra-arterial delivery.

3.6. Intranasal matrix metalloproteinase-9 pre-treatment enhanced nasally applied [¹²⁵I]-IgG access to the nervous system

Administration of tight junction modulators has been shown to improve macromolecule transport across numerous epithelial barriers throughout the body, including at nasal sites [45]. Indeed, permeation enhancement at the nasal epithelia is expected to yield higher drug and tracer levels in the lamina propria where nerve associated pathways to the brain may be most accessible [19, 20]. However, many nasal permeability enhancers cause significant nasal irritation and mucosal toxicity [46]. Matrix metalloproteinase-9 (MMP-9), a type IV collagenase and member of a large class of zinc-dependent endopeptidases, has been shown to be involved in extracellular matrix (ECM) remodeling [47] as well as tight junction modulation in part through alteration of claudin-1 [48]. Recently, we showed that intranasal MMP-9 (100 nM) can be used as a local nasal permeability enhancer [25], where claudin-1 is a prominent component of tight junctional protein complexes in the olfactory epithelium and between perineural fibroblasts of the fila olfactoria [49]. MMP-9 is naturally expressed in its active form in the olfactory mucosa where it likely plays a role in the continual replacement of epithelial cells (re-epithelialization) and olfactory sensory neurons during regular cell turnover [50].

Here, we found that intranasal [¹²⁵I]-IgG concentrations were ~5-fold higher in the olfactory bulbs^a, ~7-fold higher in the caudoputamen^b, and ~9-fold higher in the cervical spinal cord^b following intranasal MMP-9 (100 nM) pre-treatment compared to saline pre-treatment (control) (Table 4, Fig. 3; ^at(7) = 3.039, p = .019, Studenťs two-tailed t-test; ^bU = 0, p = .029, Mann-Whitney rank sum test). However, endpoint blood [¹²⁵I]-IgG concentrations were similar with MMP-9 or saline (control) pre-treatment (Table 4, Fig. 3, U = 7, p = .310 (n.s.), Mann-Whitney rank sum test). Overall, this suggests that intranasal MMP-9 pre-treatment improves nasal permeability and enhances direct delivery of [¹²⁵I]-IgG to the brain via pathways that potentially bypass the CNS barriers without increasing [¹²⁵I]-IgG systemic exposure.

Table 4.

Tissue concentrations^a (pM) of [¹²⁵I]-IgG following intranasal saline (control) vs MMP-9 (100 nM) pre-treatment.

	Intranasal (saline pre-treatment)	Intranasal (MMP-9 pre-treatment)
Blood	(pM)	(pM)
0 min	0 (4)	0 (5)
10 min	10.63 ± 0.79 (4)	18.42 ± 3.29 (5)
20 min	26.86 ± 1.48 (4)	34.72 ± 6.31 (5)
30 min	37.94 ± 2.71 (4)	50.64 ± 9.75 (5)
Blood AUC (0–30 min)	(pM*min)	(pM*min)
	562.27 ± 38.68 (4)	784.65 ± 140.63 (5)
Nervous system regions	(pM)	(pM)
ACA & MCA ^b	18.55 ± 4.44 (4) ^d	71.49 ± 21.00 (4)
Basilar & vertebral arteries ^c	26.61 ± 14.00 (5)	83.78 ± 54.36 (6)
Olfactory bulbs	4.66 ± 1.59 (5) ^e	23.86 ± 6.89 (4)
Frontal cortex	2.36 ± 0.75 (5) ^f	21.71 ± 12.62 (4)
Caudoputamen	1.01 ± 0.17 (4) ^d	7.51 ± 2.38(4)
Motor cortex	1.39 ± 0.34 (4) ^g	8.28 ± 2.00 (6)
Primary somatosensory cortex	2.13 ± 0.49 (5) ^h	5.73 ± 1.42 (4)
Midbrain	1.38 ± 0.32 (5)	4.31 ± 1.68 (3)
Posterior hippocampus	2.45 ± 0.88 (5)	8.91 ± 1.29 (4)
Medulla	1.24 ± 0.21 (5)	2.62 ± 0.88 (4)
Pons	1.82 ± 0.52 (5) ⁱ	5.85 ± 1.61 (3)
Cerebellum	1.98 ± 0.53 (5)	3.30 ± 0.58 (4)
Cervical spinal cord	1.73 ± 0.16 (4) ^d	14.82 ± 6.74 (4)
Trigeminal nerves	111.25 ± 53.21 (5)	154.66 ± 33.69 (6)
Lymph nodes	(pM)	(pM)
Deep cervical lymph nodes	151.19 ± 78.77 (5)	304.33 ± 228.74 (4)
Superficial cervical lymph nodes	81.24 ± 43.13 (5) ^f	15.05 ± 4.92 (4)
Axillary lymph nodes	4.82 ± 1.19 (5)	12.59 ± 1.08 (2)

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Blood, nervous system, and lymph node radiolabel concentrations following intranasal administration of [¹²⁵I]-IgG following saline (control) or MMP-9 (100 nM) pre-treatment. Intranasal administration of saline or MMP-9 solution was first performed drop wise (4 drops of 6 μl each) over approximately 15 min, followed by intranasal administration of a low tracer dose (50 μg) of IgG - performed drop wise (4 drops of 6 μl each) over approximately 15 min.

Data are presented as mean ± S.E.M. (n independent experiments).

anterior cerebral and middle cerebral arteries

basilar and vertebral arteries. Statistical analysis for tissue samples - saline vs MMP-9 pre-treatment: Mann-Whitney Rank sum test –

Mann-Whitney U = 0, p = .029;

t (7) = 3.039, p = .019;

Mann-Whitney U = 0, p = .016;

Mann-Whitney U = 0, p = .010; Two-tailed Studenťs t-test –

t(7) = 2.636, p = .034;

ⁱ

t(6) = 2.955, p = .025.

Fig. 3. — Fold-change in [¹²⁵I]-IgG concentrations reaching the brain following matrix metalloproteinase-9 (MMP-9) pre-administration (100 nM) compared to saline (control) pre-administration via the nasal route. Fold-change in [¹²⁵I]-IgG brain concentrations derived from data presented as mean ± S.E.M. in Table 4. Grey line indicates fold-change with respect to the intranasal saline pre-administration control data. Abbreviations: IN – intranasal; pt. – point; ACA – anterior cerebral artery; MCA – middle cerebral artery; Bas. – Basilar artery; Vert. – Vertebral arteries; PVS – perivascular space; cerv. – cervical.

3.7. Intranasal administration resulted in a widespread autoradiographic distribution of [¹²⁵I]-IgG within the CNS that corresponded with brain entry pathways

Phosphor imaging of brain sections was performed to further support our gamma counting data by providing an assessment of [¹²⁵I]-IgG distribution in the CNS with higher spatial resolution. Fig. 4 shows representative brain sections and associated schemata from animals receiving a low tracer [¹²⁵I]-IgG dose (50 μg) intranasally. The sagittal autoradiograph in Fig. 4 A1 shows that [¹²⁵I]-IgG signal was highest in the olfactory bulbs and brainstem; [¹²⁵I]-IgG signal in other brain areas mostly appeared to decrease with distance from the olfactory and trigeminal nerve brain entry points. Coronal autoradiographs showed highest [¹²⁵I]-IgG signal intensity in the ventral olfactory bulbs (Fig. 4 B1 and C1), in the vicinity of the rhinal fissure (Fig. 4 D1), and in portions of the frontal cortex (Fig. 4 D1); interestingly, [¹²⁵I]-IgG signal in the rhinal fissure and frontal cortex appeared to correspond to the approximate locations of the olfactofrontal and anterior cerebral arteries, respectively. Taken together, these autoradiographs and the gamma counting data presented above provide evidence to suggest that intranasal [¹²⁵I]-IgG gains entry to the olfactory bulbs and brainstem via olfactory and trigeminal nerve-associated pathways with further distribution hypothesized to occur in part due to transport within the perivascular spaces of cerebral blood vessels. However, the spatial re-solution of the phosphor imager (50 μm) was not sufficient to unequivocally delineate anatomical compartments such as the perivascular space. We therefore chose to further examine intranasal anti-body distribution within nasal, trigeminal, and brain tissue using fluorophore-labeled IgG coupled with high resolution laser scanning confocal microscopy.

Fig. 4. — Representative autoradiographic images of [¹²⁵I]-labeled IgG distribution in different brain regions following intranasal delivery. (A1) A sagittal rat brain section autoradiograph (approx. 1.5 mm lateral to the midline) revealed high signal in the olfactory bulbs (entry site for the olfactory nerve) along the ventral olfactory tract and in the brainstem (close to the pons – entry site for the trigeminal nerve). (A2) Schema of the sagittal rat brain section corresponding to autoradiograph A1. (B1 & C1) Coronal rat brain section autoradiographs of the olfactory bulb (approx. +7 mm bregma) showed highest signal intensity (arrows) ventrally and in the olfactory nerve layer. (B2 & C2) Schema of the coronal rat brain sections corresponding to autoradiographs B1 and C1. (D1) A coronal rat brain section autoradiograph of the frontal cortex and olfactory tract (approx. +5.6 mm bregma) showed high signal at the rhinal fissure and at the medial side of the frontal cortex. Putative signal associated with the perivascular space surrounding branches of the olfactofrontal artery and the anterior cerebral artery (asterisks). (D2) Schema of the coronal rat brain section corresponding to autoradiograph D1. Abbreviations: Olf. bulb – olfactory bulb; Cg – cingulate cortex, M – motor cortex; rf – rhinal fissure; Thal – thalamus; Hyp – Hypothalamus; HF – hippocampal formation; SC – superior colliculus; IC – inferior colliculus; Cb – cerebellum; Med – medulla; LV – lateral ventricle; 4 V – 4th ventricle; onl – olfactory nerve layer of the olfactory bulb; gl – glomerular layer of the olfactory bulb; ov – olfactory ventricle; olf. tract – olfactory tract; ACA – anterior cerebral artery; OFA – olfactofrontal artery. Rat brain section schemas were adapted from the Rat Brain Atlas [40].

3.8. Imaging of fluorophore-labeled IgG distribution following intranasal administration revealed olfactory and trigeminal brain entry pathways and further distribution within the CNS via perivascular spaces

We used intranasal MMP-9 (100 nM) pre-treatment to enhance intranasal delivery of a high dose (0.7 mg) of Alexa Fluor 488 labeled IgG (AF488-IgG) to maximize signal-to-noise and allow more detailed study of IgG distribution in nasal, trigeminal, and CNS tissue at higher resolution using epifluorescence microscopy and laser scanning confocal microscopy. Figs. 5–7 show representative images from animals 30 min post intranasal administration. Low magnification ex vivo imaging of the nasal cavity showed high AF488-IgG signal in both the nasal respiratory and olfactory mucosae (Fig. 5A and B). High magnification confocal imaging of sections from the olfactory epithelium, a pseudostratified columnar structure containing olfactory sensory neurons, sustentacular (supporting) cells, basal cells and occasional Bowman's glands [19], showed AF488-IgG signal concentrated at the epithelial surface but also significant accumulation within the epithelium and in the underlying lamina propria (Fig. 5C). AF488-IgG signal could be clearly seen surrounding PGP 9.5-positive (neuronal marker) bundles of olfactory axons, arranged in olfactory fascicles [51] within the lamina propria (Fig. 5E, circle), as well as surrounding luminal structures resembling either blood or lymphatic vessels (Fig. 5E, asterisk). The observed signal in the olfactory lamina propria suggested intranasal AF488-IgG accessed multiple compartments: (i) perineural localization in the vicinity of olfactory nerve fibroblasts making up the outer boundary of nerve fascicles, (ii) lesser perineural signal within the interior of nerve fascicles in the vicinity of olfactory ensheathing cells and olfactory axons, and (iii) perivascular localization around blood vessels and lymphatics (shown schematically in Fig. 5E). High magnification confocal imaging of sections from the nasal respiratory mucosa also showed AF488-IgG signal concentrated at the epithelial surface with significant accumulation in the underlying lamina propria (Fig. 5D).

Fig. 5. — Fluorescence imaging of AF488-IgG distribution in the nasal mucosa following intranasal delivery. (A) Low magnification *ex vivo* imaging revealed AF488-IgG exposure to the olfactory epithelium (OE) and respiratory epithelium (RE) at 30 min post administration. (B) Schema showing the architecture and epithelial types lining the lateral wall of the rat nasal cavity; figure adapted from [31], based on [53, 54]. (C) A coronal section of the olfactory mucosa showed high AF488-IgG signal at the epithelial surface and lesser but still significant signal within the the epithelium and in the underlying lamina propria. The asterisk indicates IgG accessing a putative Bowman's gland, a simple tubular structure that traverses through the basal lamina and olfactory epithelium to open into the nasal passage [54]. (D) A coronal section of the respiratory mucosa showed AF488-IgG signal at the epithelial surface, within the epithelium, and in the underlying lamina propria. (E) A coronal section of the olfactory mucosa showed high AF488-IgG signal at the epithelial surfaces facing the nasal passage, within the olfactory epithelium, and within the lamina propria. In the lamina propria, AF488-IgG was deposited around putative blood or lymphatic vessels (asterisk) and bundles of olfactory axons arranged as fascicles (PGP 9.5-positive). The dashed white circle shows AF488-IgG distribution within an individual nerve fascicle and surrounding vessels, represented schematically at far right. Highest AF488-IgG levels were typically evident perineurally at the periphery of fascicles (lined by olfactory nerve fibroblasts and basal lamina, as indicated [51] and in putative perilymphatic and perivascular compartments. Lower AF488-IgG signal was also often observed between olfactory ensheathing cells and individual axons within each fascicle. Neuronal cell bodies and axons were labeled with an anti-PGP 9.5 antibody; Cell nuclei were labeled using DAPI. A: *ex-vivo* imaging; *C-E*: laser scanning confocal microscopy. Representative images from n = 3 rats. Abbreviations: CP – cribriform plate; ET – ethmoturbinate; NT – naso-turbinate; MT – maxilloturbinate; NP – nasopharynx; NALT – nasal-associated lymphoid tissue; LP – lamina propria; OE – olfactory epithelium; RE – respiratory epithelium; OEC – olfactory ensheathing cell; PGP 9.5 – protein gene product 9.5; DAPI – 4′,6-diamidino-2-phenylindole. Nasal epithelia and lamina propria layers are delineated by straight white dashed lines in C-E.

Fig. 7. — Fluorescence imaging of AF488-IgG distribution in the brain following intranasal delivery. Intranasal delivery of AF488-IgG following 100 nM matrix metalloproteinase-9 (MMP-9) intranasal pre-treatment resulted in AF488-IgG signal in the olfactory bulb nerve layer (*A-D*) as well as along perivascular spaces of cerebral blood vessels (*B-D*). AF488-IgG signal in the perivascular compartment of the anterior cerebral circulation was observed at (E) the rhinal fissure and (F) at the longitudinal fissure between the two brain hemispheres. (*G, H*) Perivascular AF488-IgG signal in the rhinal fissure was localized outside the smooth muscle layer of vessels identified as putative arteries based on morphology. *A-H*: laser scanning confocal microscopy. Vascular endothelial cells were labeled with an anti-RECA-1 antibody; astrocytes were labeled with an anti-GFAP antibody; smooth muscle cells were labeled with an anti-α-SMA antibody; cell nuclei were labeled using DAPI. Representative images from n = 3 rats. Abbreviations: gl – glomerular layer of the olfactory bulb; onl – olfactory nerve layer of the olfactory bulb; pvs – perivascular space; RECA-1 – rat endothelial cell antigen-1; GFAP – glial fibrillary acidic protein; α-SMA – alpha smooth muscle actin; DAPI: 4,6-diamidino-2-phenylindole.

Low magnification imaging of the base of the skull (schema in Fig. 1B) showed that intranasal AF488-IgG resulted in high signal in the vicinity of the ophthalmic (V1) and maxillary (V2) branches of the trigeminal nerve with no signal apparent in the vicinity of the mandibular branch (V3) (Fig. 6A); since V1 and V2, but not V3, innervate the nasal mucosa [19], this result was consistent with trigeminal nerve anatomical relationships with the nasal cavity. A section parallel to the long axis of the V1/V2 segments of the trigeminal nerve revealed significant AF488-IgG signal both within and around nerve (Fig. 6B); a schematic of the anatomical relationships for a transverse section through the nerve (dashed line) is also shown. Like most peripheral nerves, the trigeminal nerve has three connective tissue sheaths: an outermost epineurium, a middle perineurium, and an innermost endoneurium [52]. The innermost endoneurium contains axons running parallel to the long axis of the nerve, blood vessels with a poorly developed smooth muscle layer, supporting cells, and accompanying extracellular matrix. The perineurium contains flattened fibroblast-like cells with tight junctions that surround groups of axons and other endoneurial components to create a nerve fascicle. The outermost epineurium is a loose connective tissue sheath containing fibroblasts, collagen, and blood vessels that surrounds the entire nerve. AF488-IgG signal was prominent in the outermost epineurium (Fig. 6B, asterisk) as well as interior compartments of the nerve (Fig. 6B, double asterisk). High magnification images of the endoneurial layer (Fig. 6C and D) showed prominent high levels of AF488-IgG signal in the perivascular spaces of endoneurial blood vessels with lower diffuse AF488-IgG signal surrounding axons and other endoneurial components. Taken together, the results of our imaging of the nasal mucosa and innervating trigeminal nerves following intranasal AF488-IgG (Figs. 5 and 6) suggested AF488-IgG accessed both perineural and perivascular compartments associated with both olfactory and trigeminal nerve components.

Fig. 6. — Fluorescence imaging of AF488-IgG distribution in the trigeminal nerves following intranasal delivery. (A) A view of the skull base 30 min following intranasal delivery showed AF488-IgG signal predominantly in the vicinty of V1 (ophthalmic) and V2 (maxillary) branches of the trigeminal nerve. (B) AF488-IgG signal was evident around and within V1 and V2 branches in a section of the trigeminal nerve cut parallel to its long axis. A schematic of the anatomical relationships for a transverse section through the nerve (dashed line) indicates how the connective tissue sheaths are typically arranged as epineurium (Ep), perineurium (P), and endoneurium (En). (*C, D*) Imaging of the endoneurium showed high AF488-IgG signal within the perivascular space of blood vessels and lower diffuse AF488-IgG signal between axons and occasionally within neuronal cell bodies. Vascular endothelial cells were labeled with an anti-RECA-1 antibody; neuronal cell bodies and axons were labeled with an anti-PGP 9.5 antibody; Cell nuclei were labeled using DAPI. A: *ex vivo* imaging; *B-D*: laser scanning confocal microscopy. Representative images from n = 3 rats. Abbreviations: pvs – perivascular space; RECA-1 – rat endothelial cell antigen-1; PGP 9.5 – protein gene product 9.5; DAPI – 4,6-diamidino-2-phenylindole.

Remarkably, intranasal application of AF488-IgG following MMP-9 pretreatment yielded sufficient signal for imaging to shed further light on its brain distribution beyond the olfactory and trigeminal brain entry pathways, despite obtaining only low nanomolar IgG levels in regions exhibiting the best delivery (e.g., the olfactory bulbs and the perivascular spaces of certain cerebral blood vessels; see Tables 2–4). As we have discussed previously [25], fluorescence-based methods have lower sensitivity than the radiolabel-based methods employed above so our ability to successfully image AF488-IgG distribution across different brain regions was constrained by low signal-to-noise at most CNS sites, despite our use of a highly concentrated AF488-IgG solution approaching the solubility limit and intranasal pretreatment with a nasal epithelial permeability enhancer (MMP-9). Nevertheless, AF488-IgG signal was often observed at the level of the olfactory bulbs, with highest signal in the olfactory nerve layer (Fig. 7A–D) as well as along perivascular spaces (Fig. 7B–D) and lower diffuse signal observed within the parenchyma (Fig. 7A–D). AF488-IgG signal in the perivascular compartment of the anterior cerebral circulation was also evident at the rhinal fissure (Fig. 7E) and around the longitudinal fissure between the two cerebral hemispheres (Fig. 7F). Perivascular AF488-IgG signal in the rhinal fissure was localized outside the smooth muscle layer (Fig. 7G) and endothelial cell layer (Fig. 7H), in agreement with a predominantly adventitial perivascular distribution that has been demonstrated for fluorescently labeled IgG and other large macromolecules administered by different routes (e.g., intrathecal; [34]). Supplementary Fig. 1 further demonstrates that the perivascular AF488-IgG signal observed in the brain following intranasal administration is not endogenous autofluorescence (e.g., from the internal elastic lamina); only a select subset of vessels exhibited this perivascular fluorescent signal and its nature was similar to previously published work that has examined perivascular distribution of fluorophore-labeled IgG following intrathecal administration [34].

4. Discussion

Our study resulted in three major findings. First, we showed that for IgG doses resulting in similar endpoint blood concentrations, intranasal delivery resulted in higher IgG concentrations within the CNS and a better dose-response than intra-arterial delivery. Second, we showed that IgG can rapidly reach the CNS via unique perineural and perivascular pathways that potentially bypass the BBB and BCSFBs following intranasal delivery. Third, intranasal IgG delivery to the CNS can be enhanced by modulating nasal epithelial permeability.

Highest [¹²⁵I]-IgG concentrations across all intranasal doses were consistently associated with the olfactory bulbs, the ophthalmic and maxillary branches of the trigeminal nerves which innervate the nasal mucosa, and the perivascular spaces (PVS) of major cerebral arteries. This suggests that intranasal [¹²⁵I]-IgG entered and distributed within the brain via pathways associated with the olfactory nerves, trigeminal nerves, and PVS, as reported for other proteins and dextrans following intranasal delivery in rodents [23, 25] and non-human primates [24]. Importantly, [¹²⁵I]-IgG brain levels were significantly higher following all intranasal doses compared to corresponding endpoint blood concentration matched intra-arterial doses, consistent with delivery of [¹²⁵I]-IgG to the CNS via pathways unique to intranasal delivery that bypass the BBB and BCSFBs. Additionally, fold changes in [¹²⁵I]-IgG concentrations within several brain regions were either similar to or exceeded the fold change in endpoint [¹²⁵I]-IgG blood levels for 20-fold and 50-fold higher intranasal doses. Overall, [¹²⁵I]-IgG brain concentrations following intranasal administration were in the low to mid picomolar range for the low tracer dose and scaled up to the low nanomolar range for the 20-fold higher and 50-fold higher doses; this range approaches and even exceeds estimates of antibody concentrations likely to be necessary for therapeutic effects (e.g., crenezumab, an anti-β-amyloid IgG, has been reported efficacious over the picomolar range [55], even at concentrations as low as ~ 20 pM (EC50) [56]). In contrast, [¹²⁵I]-IgG concentrations within all nervous tissue sampled except the trigeminal nerves did not appreciably scale up with increasing intra-arterial doses and were below the levels likely to be needed for therapeutic IgG effects. Changes in intra-arterially administered [¹²⁵I]-IgG concentrations in the trigeminal nerves with increasing doses are likely explained by the relatively high permeability of blood vessels within the outermost trigeminal nerve connective tissue (epineurium) [57]. It should be noted that although intranasal doses resulted in significantly higher [¹²⁵I]-IgG concentrations within the CNS compared to intra-arterial doses that produced similar end-point blood [¹²⁵I]-IgG levels, these concentrations were still low and indicate that only a small fraction of intranasally administered [¹²⁵I]-IgG accessed the CNS at an early time point (30 min) following a single acute dose. Repeated intranasal dosing with smaller doses may be considered to achieve similar or higher concentrations.

Imaging of the distribution of intranasally administered AF488-IgG in the nasal mucosae confirmed its ability to migrate through the nasal epithelia to the lamina propria via paracellular and/or transcellular transport pathways. Tight junction (TJ) protein complexes in epithelia form a key physiological barrier for paracellular transport of hydrophilic macromolecules [58]. However, exposure to the external environment and frequent cell turnover results in continuous nasal epithelial reorganization and possible TJ loosening [59]. Paracellular transport across the nasal epithelia may also be enhanced by intranasal MMP-9, a physiologic and local TJ modulator [25]. Finally, intranasally administered IgG may undergo transcellular transport across the nasal epithelia via an FcRn-dependent mechanism [60, 61].

Once intranasally applied IgG accesses the lamina propria, it may be (i) systemically absorbed by nasal blood vessels, (ii) absorbed by nasal lymphatics and transported to the cervical lymph nodes, and/or (iii) available to perineural and perivascular channels associated with the olfactory and trigeminal nerves for brain entry. Our data confirmed intranasal [¹²⁵I]-IgG resulted in delivery to the systemic circulation (blood levels increased with dose and over time), local lymphatics (high exposure was measured within cervical lymph nodes), and multiple brain areas. We previously demonstrated that intranasal MMP-9 pre-administration can enhance brain delivery of intranasally administered 10 kDa dextran (dex10); here, we observed a similar enhancement of [¹²⁵I]-IgG brain entry with intranasal MMP-9 pretreatment. It might be expected that MMP-9 facilitation of intranasal dex10 and [¹²⁵I]-IgG access to the lamina propria would be accompanied by elevated blood levels; however, neither dex10 [25] nor [¹²⁵I]-IgG levels increased in blood samples with intranasal MMP-9 pretreatment. Consideration of the factors that regulate systemic absorption from the nasal lamina propria may provide a possible explanation for these results. We recently reported that the capillary density and relative vascular permeability are significantly higher in the rodent nasal respiratory mucosa than the olfactory mucosa [31], consistent with the former being the main site for systemic absorption of intranasally administered substances. Interestingly, substance P immunoreactive trigeminal nerve fibers are found in association with arterioles and venules within the nasal respiratory lamina propria [62], where they facilitate vasodilation [63]. Since MMP-9 can degrade substance P [64], intranasal MMP-9 may indirectly lead to vasoconstriction in the nasal respiratory mucosa, attenuating dex10 and [¹²⁵I]-IgG systemic absorption. Nevertheless, we show that intranasal administration can lead to significant IgG blood exposure that may be important for certain responses, e.g., peripheral sink effects for anti-amyloid antibodies in Alzheimer's disease [65]. Intranasal IgG targeting to the cervical lymph nodes, as demonstrated here, may be particularly relevant for mounting an immune response to CNS antigens [66, 67] or to target primary CNS tumor metastases typically localized in the cervical lymph nodes [68, 69].

Any IgG within the nasal lamina propria that escapes absorption into the local blood and lymphatic vessels may access extracellular compartments associated with the olfactory and trigeminal nerves, as suggested by other studies [19, 23–25]. Localization of intranasal AF488-IgG in the vicinity of olfactory axon bundles within the nasal lamina propria, within connective tissue sheaths of the trigeminal nerves, and in the PVS of blood vessels in the nasal mucosae and trigeminal nerves was therefore expected.

Substances injected into the CSF and brain parenchyma have been shown to drain along the perineural spaces of olfactory nerves [49, 51] and, from there, the cervical lymph nodes may be reached via nasal lymphatics [70–73]; these interconnections may potentially be bidirectional and provide a route for intranasally administered substances to reach the subarachnoid space CSF and brain [23, 74, 75]. Our tracer dosing results showed endpoint CSF [¹²⁵I]-IgG concentrations were significantly higher following intranasal than following matched intra-arterial administration; CSF [¹²⁵I]-IgG also increased significantly over time only following intranasal dosing. The superiority of the nasal route over systemic routes in targeting macromolecules to the CSF has been reported previously for other macromolecule tracers like dextrans [76], although some intranasally administered macromolecules (e.g., 8 kDa insulin-like growth factor-I) have not appeared to access the CSF despite pronounced delivery to the brain [23]. Here, endpoint CSF [¹²⁵I]-IgG concentrations were ~ 2-fold to 30-fold lower than concentrations measured in brain tissue 30 min following intranasal delivery; this suggests [¹²⁵I]-IgG access to brain sites likely occurred without CSF distribution first and is in keeping with CSF concentrations not always accurately reflecting brain target site levels [77]. Sakane and colleagues suggested that CSF access following intranasal delivery is size-dependent, decreasing with increasing size for dextrans spanning 4–20 kDa and not detectable for 40 kDa dextran [76]. Subsequent work has shown that intranasally applied low molecular weight peptides, e.g., oxytocin (1 kDa), readily access the CSF of rodents [78, 79], monkeys [80], and humans [81]. There is increasing evidence for a size-dependent sieving process governing molecule transfer into the CSF across lining cells of nerves and leptomeningeal vessels [34, 43]. The ability of intranasally administered IgG to access the CSF at limited but detectable levels despite its large size may in part be due to unique physio-logical roles for IgG in immune surveillance [34] and FcRn-dependent mechanisms [82] that could actively transport IgG into the CSF from olfactory and/or trigeminal nerve-associated compartments as they transit the subarachnoid space to reach the brain.

We speculate that IgG transfer between perineural compartments of the olfactory and trigeminal brain entry pathways and PVS in the brain and spinal cord may be linked via the subpial space [83, 84]. Further work is needed to better clarify fluid exchange relationships between nerves, brain, PVS, and CSF, particularly after intranasal drug delivery. Nevertheless, our current and prior studies [25, 34] emphasize that distribution within perivascular pathways may be a key determinant of widespread brain delivery by the intranasal route as well as other central routes of administration. We have shown here that such mechanisms may allow therapeutically relevant levels of intranasally delivered IgG to access the brain, potentially offering a non-invasive approach for CNS immunotherapies.

5. Conclusions

The delivery of antibody-based therapeutics via the systemic route to the central nervous system (CNS) remains a challenge due to the presence of the blood-brain barrier (BBB) and blood-cerebrospinal fluid barriers (BCSFBs). Here, we showed it is possible to rapidly achieve therapeutically relevant concentrations of immunoglobulin G (IgG) in the brain following intranasal delivery. Intranasal administration resulted in higher CNS IgG concentrations and superior dose-response characteristics compared to systemic administration across several doses resulting in similar endpoint blood levels via either route. Intranasal IgG accessed the CNS via unique extracellular perineural and perivascular pathways that bypassed the BBB and BCSFBs. Overall, our results support the potential of the intranasal route as a means to deliver IgG to the CNS.

Supplementary Material

NIHMS1504903-supplement-1.eps^{(8MB, eps)}

NIHMS1504903-supplement-2.eps^{(1.1MB, eps)}

NIHMS1504903-supplement-3.docx^{(2.1MB, docx)}

Acknowledgements

This work was generously supported by the Michael J. Fox Foundation for Parkinson's Research (UW Reference # MSN189990), the Clinical and Translational Science Award program administered through the NIH National Center for Advancing Translational Sciences (NIH UL1TR000427 and KL2TE000428), the Wisconsin Alumni Research Foundation Accelerator Program, the University of Wisconsin-Madison School of Pharmacy, the Graduate School at the University of Wisconsin-Madison, fellowships through the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1256259 (MEP), NIH fellowships (NRSA T32 EBO11434 - MEP), Parkinson's Foundation-American Parkinson's Disease Association Summer Student Fellowship (PF-APDA-SFW-1730 - SB), the Hilldale Undergraduate Research Fellowship from the University of Wisconsin-Madison (SB), and the Howard Hughes Medical Institute Precollege and Undergraduate Science Education Program grant to Macalester College (GG). Laser scanning confocal microscopy was performed with training and guidance from Dr. Michael Taylor (Nikon A1R) and Dr. Arash Bashirullah (Olympus FV1000) at the University of Wisconsin-Madison School of Pharmacy.

Footnotes

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jconrel.2018.08.006.

References

[1].Yu CY, Ng G, Liao P, Therapeutic antibodies in stroke, Transl. Stroke Res 4 (2013) 477–483. [DOI] [PMC free article] [PubMed] [Google Scholar]
[2].Lannfelt L, Möller C, Basun H, Osswald G, Sehlin D, Satlin A, Logovinsky V, Gellerfors P, Perspectives on future Alzheimer therapies: amyloid-β protofibrils - a new target for immunotherapy with BAN2401 in Alzheimer's disease, Alzheimers Res. Ther 6 (2014) 16. [DOI] [PMC free article] [PubMed] [Google Scholar]
[3].George S, Brundin P, Immunotherapy in Parkinson's disease: micromanaging alpha-Synuclein aggregation, J. Parkinsons Dis 5 (2015) 413–424. [DOI] [PMC free article] [PubMed] [Google Scholar]
[4].Chacko AM, Li C, Pryma DA, Brem S, Coukos G, Muzykantov V, Targeted delivery of antibody-based therapeutic and imaging agents to CNS tumors: crossing the blood-brain barrier divide, Expert Opin. Drug Deliv 10 (2013) 907–926. [DOI] [PMC free article] [PubMed] [Google Scholar]
[5].Rommer PS, Dudesek A, Stüve O, Zettl UK, Monoclonal antibodies in treatment of multiple sclerosis, Clin. Exp. Immunol 175 (2014) 373–384. [DOI] [PMC free article] [PubMed] [Google Scholar]
[6].Hammarlund-Udenaes M, de Lange E, Thorne R, Drug Delivery to the Brain, 1 ed., AAPSPress. Springer, 2014. [Google Scholar]
[7].Reese TS, Karnovsky MJ, Fine structural localization of a blood-brain barrier to exogenous peroxidase, J. Cell Biol 34 (1967) 207–217. [DOI] [PMC free article] [PubMed] [Google Scholar]
[8].Becker NH, Novikoff AB, Zimmerman HM, Fine structure observations of the uptake of intravenously injected peroxidase by the rat choroid plexus, J. Histochem. Cytochem 15 (1967) 160–165. [DOI] [PubMed] [Google Scholar]
[9].Nabeshima S, Reese TS, Landis DM, Brightman MW, Junctions in the meninges and marginal glia, J. Comp. Neurol 164 (1975) 127–169. [DOI] [PubMed] [Google Scholar]
[10].Poduslo JF, Curran GL, Berg CT, Macromolecular permeability across the blood-nerve and blood-brain barriers, Proc. Natl. Acad. Sci. U. S. A 91 (1994) 5705–5709. [DOI] [PMC free article] [PubMed] [Google Scholar]
[11].Davson H, Segal M, Physiology of the CSF and Blood-brain Barriers, CRC Press, 1995. [Google Scholar]
[12].Balin BJ, Broadwell RD, Salcman M, El-Kalliny M, Avenues for entry of peripherally administered protein to the central nervous system in mouse, rat, and squirrel monkey, J. Comp. Neurol 251 (1986) 260–280. [DOI] [PubMed] [Google Scholar]
[13].Broadwell RD, Sofroniew MV, Serum proteins bypass the blood-brain fluid barriers for extracellular entry to the central nervous system, Exp. Neurol 120 (1993) 245–263. [DOI] [PubMed] [Google Scholar]
[14].Banks WA, Terrell B, Farr SA, Robinson SM, Nonaka N, Morley JE, Passage of amyloid beta protein antibody across the blood-brain barrier in a mouse model of Alzheimer's disease, Peptides 23 (2002) 2223–2226. [DOI] [PubMed] [Google Scholar]
[15].Wang Q, Delva L, Weinreb PH, Pepinsky RB, Graham D, Veizaj E, Cheung AE, Chen W, Nestorov I, Rohde E, Caputo R, Kuesters GM, Bohnert T, Gan LS, Monoclonal antibody exposure in rat and cynomolgus monkey cerebrospinal fluid following systemic administration, Fluids Barriers CNS 15 (2018) 10. [DOI] [PMC free article] [PubMed] [Google Scholar]
[16].Levites Y, Smithson LA, Price RW, Dakin RS, Yuan B, Sierks MR, Kim J, McGowan E, Reed DK, Rosenberry TL, Das P, Golde TE, Insights into the mechanisms of action of anti-Abeta antibodies in Alzheimer's disease mouse models, FASEB J. 20 (2006) 2576–2578. [DOI] [PubMed] [Google Scholar]
[17].St-Amour I, Paré I, Alata W, Coulombe K, Ringuette-Goulet C, Drouin-Ouellet J, Vandal M, Soulet D, Bazin R, Calon F, Brain bioavailability of human intravenous immunoglobulin and its transport through the murine blood-brain barrier, J. Cereb. Blood Flow Metab 33 (2013) 1983–1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
[18].Petereit HF, Rubbert-Roth A, Rituximab levels in cerebrospinal fluid of patients with neurological autoimmune disorders, Mult. Scler 15 (2009) 189–192. [DOI] [PubMed] [Google Scholar]
[19].Lochhead JJ, Thorne RG, Intranasal delivery of biologics to the central nervous system, Adv. Drug Deliv. Rev 64 (2012) 614–628. [DOI] [PubMed] [Google Scholar]
[20].Lochhead J, Thorne R, Intranasal drug delivery to the brain, in: Hammarlund-Udenaes D, Lange Thorne (Eds.), Drug Delivery to the Brain - Physiological concepts, Methodologies and Approaches, Springer, 2014, pp. 401–431. [Google Scholar]
[21].Dhuria SV, Hanson LR, Frey WH, Intranasal delivery to the central nervous system: mechanisms and experimental considerations, J. Pharm. Sci 99 (2010) 1654–1673. [DOI] [PubMed] [Google Scholar]
[22].Illum L, Nasal drug delivery - recent developments and future prospects, J. Control. Release 161 (2012) 254–263. [DOI] [PubMed] [Google Scholar]
[23].Thorne RG, Pronk GJ, Padmanabhan V, Frey WH, Delivery of insulin-like growth factor-I to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration, Neuroscience 127 (2004) 481–496. [DOI] [PubMed] [Google Scholar]
[24].Thorne RG, Hanson LR, Ross TM, Tung D, Frey WH, Delivery of interferon-beta to the monkey nervous system following intranasal administration, Neuroscience 152 (2008) 785–797. [DOI] [PubMed] [Google Scholar]
[25].Lochhead JJ, Wolak DJ, Pizzo ME, Thorne RG, Rapid transport within cerebral perivascular spaces underlies widespread tracer distribution in the brain after intranasal administration, J. Cereb. Blood Flow Metab 35 (2015) 371–381. [DOI] [PMC free article] [PubMed] [Google Scholar]
[26].Asgari M, Zélicourt D, Kurtcuoglu V, Glymphatic solute transport does not require bulk flow, Sci Rep 6 (2016) 38635. [DOI] [PMC free article] [PubMed] [Google Scholar]
[27].Kolobov VV, Davydova TV, Zakharova IA, Gorbatov VIU, Fomina VG, Repressional effects of the glutamate antibodies on expression of Dffb gene in the brain of rats with experimental Alzheimer's disease, Mol. Biol. (Mosk.) 46 (2012) 757–765. [PubMed] [Google Scholar]
[28].Kolobov VV, Zakharova IA, Fomina VG, Gorbatov VY, Davydova TV, Effect of antibodies to glutamate on caspase-3 activity in brain structures of rats with experimental Alzheimer's disease, Bull. Exp. Biol. Med 154 (2013) 425–427. [DOI] [PubMed] [Google Scholar]
[29].Chauhan MB, Chauhan NB, Brain uptake of neurotherapeutics after intranasal versus intraperitoneal delivery in mice, J. Neurol. Neurosurg 2 (2015). [PMC free article] [PubMed] [Google Scholar]
[30].Cattepoel S, Hanenberg M, Kulic L, Nitsch RM, Chronic intranasal treatment with an anti-Aβ(30–42) scFv antibody ameliorates amyloid pathology in a trans-genic mouse model of Alzheimer's disease, PLoS One 6 (2011) e18296. [DOI] [PMC free article] [PubMed] [Google Scholar]
[31].Kumar NN, Gautam M, Lochhead JJ, Wolak DJ, Ithapu V, Singh V, Thorne RG, Relative vascular permeability and vascularity across different regions of the rat nasal mucosa: implications for nasal physiology and drug delivery, Sci. Rep 6 (2016) 31732. [DOI] [PMC free article] [PubMed] [Google Scholar]
[32].Frey W, Delivery of 125I-NGF to the brain via the olfactory route, Drug Deliv. 4 (1997) 87–92. [Google Scholar]
[33].Guide for the care and use of Laboratory Animals, The National Academies Press, Washington, D.C., 2011.
[34].Pizzo ME, Wolak DJ, Kumar NN, Brunette E, Brunnquell CL, Hannocks MJ, Abbott NJ, Meyerand ME, Sorokin L, Stanimirovic DB, Thorne RG, Intrathecal antibody distribution in the rat brain: surface diffusion, perivascular transport, and osmotic enhancement of delivery, J. Physiol 596 (3) (2018) 445–475. [DOI] [PMC free article] [PubMed] [Google Scholar]
[35].Schmitt C, Strazielle N, Ghersi-Egea JF, Brain leukocyte infiltration initiated by peripheral inflammation or experimental autoimmune encephalomyelitis occurs through pathways connected to the CSF-filled compartments of the forebrain and midbrain, J. Neuroinflammation 9 (2012) 187. [DOI] [PMC free article] [PubMed] [Google Scholar]
[36].Fraker PJ, Speck JC, Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril, Biochem. Biophys. Res. Commun 80 (1978) 849–857. [DOI] [PubMed] [Google Scholar]
[37].Paxinos G, The Rat Nervous System, 4th ed., Elsevier, London, 2015. [Google Scholar]
[38].Thorne R, Primer on central nervous system structure/function and the vasculature, ventricular system, and fluids of the brain, in: Hammarlund-Udenaes D, Lange Thorne (Eds.), Drug Delivery to the Brain - Physiological Concepts, Methodologies and Approaches, Springer, 2014, pp. 685–707. [Google Scholar]
[39].Palkovits M, Brownstein MJ, Maps and Guide to Microdissection of the Rat Brain, Elsevier, New York, 1988. [Google Scholar]
[40].Paxinos G, Watson C, The Rat Brain in Stereotaxic Co-ordinates, 6th ed., Elsevier Inc., 2007. [Google Scholar]
[41].Bleys RL, Groen GJ, Hommersom RF, Neural connections in and around the cavernous sinus in rat, with special reference to cerebrovascular innervation, J. Comp. Neurol 369 (1996) 277–291. [DOI] [PubMed] [Google Scholar]
[42].Aspelund A, Antila S, Proulx ST, Karlsen TV, Karaman S, Detmar M, Wiig H, Alitalo K, A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules, J. Exp. Med 212 (2015) 991–999. [DOI] [PMC free article] [PubMed] [Google Scholar]
[43].Abbott NJ, Pizzo ME, Preston JE, Janigro D, Thorne RG, The role of brain barriers in fluid movement in the CNS: is there a 'glymphatic' system? Acta Neuropathol. 135 (2018) 387–407. [DOI] [PubMed] [Google Scholar]
[44].Bien-Ly N, Boswell CA, Jeet S, Beach TG, Hoyte K, Luk W, Shihadeh V, Ulufatu S, Foreman O, Lu Y, Devoss J, van der Brug M, Watts RJ, Lack of widespread BBB disruption in Alzheimer's disease models: focus on therapeutic antibodies, Neuron 88 (2015) 289–297. [DOI] [PubMed] [Google Scholar]
[45].Deli MA, Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery, Biochim. Biophys. Acta 1788 (2009) 892–910. [DOI] [PubMed] [Google Scholar]
[46].Marttin E, Verhoef JC, Cullander C, Romeijn SG, Nagelkerke JF, Merkus FW, Confocal laser scanning microscopic visualization of the transport of dextrans after nasal administration to rats: effects of absorption enhancers, Pharm. Res 14 (1997) 631–637. [DOI] [PubMed] [Google Scholar]
[47].Yabluchanskiy A, Ma Y, Iyer RP, Hall ME, Lindsey ML, Matrix metalloprotei-nase-9: many shades of function in cardiovascular disease, Physiology (Bethesda) 28 (2013) 391–403. [DOI] [PMC free article] [PubMed] [Google Scholar]
[48].Hackel D, Krug SM, Sauer RS, Mousa SA, Böcker A, Pflücke D, Wrede EJ, Kistner K, Hoffmann T, Niedermirtl B, Sommer C, Bloch L, Huber O, Blasig IE, Amasheh S, Reeh PW, Fromm M, Brack A, Rittner HL, Transient opening of the perineurial barrier for analgesic drug delivery, Proc. Natl. Acad. Sci. U. S. A 109 (2012) E2018–E2027. [DOI] [PMC free article] [PubMed] [Google Scholar]
[49].Wolburg H, Wolburg-Buchholz K, Sam H, Horvát S, Deli MA, Mack AF, Epithelial and endothelial barriers in the olfactory region of the nasal cavity of the rat, Histochem. Cell Biol 130 (2008) 127–140. [DOI] [PubMed] [Google Scholar]
[50].Gueye Y, Ferhat L, Sbai O, Bianco J, Ould-Yahoui A, Bernard A, Charrat E, Chauvin JP, Risso JJ, Féron F, Rivera S, Khrestchatisky M, Trafficking and secretion of matrix metalloproteinase-2 in olfactory ensheathing glial cells: a role in cell migration? Glia 59 (2011) 750–770. [DOI] [PubMed] [Google Scholar]
[51].Li Y, Field PM, Raisman G, Olfactory ensheathing cells and olfactory nerve fibroblasts maintain continuous open channels for regrowth of olfactory nerve fibres, Glia 52 (2005) 245–251. [DOI] [PubMed] [Google Scholar]
[52].Wigley C, Nervous system, 40th ed., Churchill Livingstone Elsevier, Spain, 2008. [Google Scholar]
[53].Mery S, Gross EA, Joyner DR, Godo M, Morgan KT, Nasal diagrams: a tool for recording the distribution of nasal lesions in rats and mice, Toxicol. Pathol 22 (1994) 353–372. [DOI] [PubMed] [Google Scholar]
[54].Harkema JR, Carey SA, Wagner JG, The nose revisited: a brief review of the comparative structure, function, and toxicologic pathology of the nasal epithelium, Toxicol. Pathol 34 (2006) 252–269. [DOI] [PubMed] [Google Scholar]
[55].Adolfsson O, Pihlgren M, Toni N, Varisco Y, Buccarello AL, Antoniello K, Lohmann S, Piorkowska K, Gafner V, Atwal JK, Maloney J, Chen M, Gogineni A, Weimer RM, Mortensen DL, Friesenhahn M, Ho C, Paul R, Pfeifer A, Muhs A, Watts RJ, An effector-reduced anti-β-amyloid (Aβ) antibody with unique aβ binding properties promotes neuroprotection and glial engulfment of Aβ, J. Neurosci 32 (2012) 9677–9689. [DOI] [PMC free article] [PubMed] [Google Scholar]
[56].Fuller JP, Stavenhagen JB, Christensen S, Kartberg F, Glennie MJ, Teeling JL, Comparing the efficacy and neuroinflammatory potential of three anti-abeta anti-bodies, Acta Neuropathol. 130 (2015) 699–711. [DOI] [PMC free article] [PubMed] [Google Scholar]
[57].Arvidsson B, Kristensson K, Olsson Y, Vascular permeability to fluorescent protein tracer in trigeminal nerve and gasserian ganglion, Acta Neuropathol. 26 (1973) 199–205. [DOI] [PubMed] [Google Scholar]
[58].Matter K, Balda MS, SnapShot: epithelial tight junctions, Cell 157 (2014) 992. [DOI] [PubMed] [Google Scholar]
[59].Altner H, Altner-Kolnberger I, Freeze-fracture and tracer experiments on the permeability of the zonulae occludentes in the olfactory mucosa of vertebrates, Cell Tissue Res. 154 (1974) 51–59. [DOI] [PubMed] [Google Scholar]
[60].Ye L, Zeng R, Bai Y, Roopenian DC, Zhu X, Efficient mucosal vaccination mediated by the neonatal Fc receptor, Nat. Biotechnol 29 (2011) 158–163. [DOI] [PMC free article] [PubMed] [Google Scholar]
[61].Heidl S, Ellinger I, Niederberger V, Waltl EE, Fuchs R, Localization of the human neonatal Fc receptor (FcRn) in human nasal epithelium, Protoplasma 253 (2016) 1557–1564. [DOI] [PubMed] [Google Scholar]
[62].Finger T, Silver W, The Neurobiology of Taste and Smell, 2 ed., (1987). [Google Scholar]
[63].Bertrand C, Geppetti P, Baker J, Petersson G, Piedimonte G, Nadel JA, Role of peptidases and NK1 receptors in vascular extravasation induced by bradykinin in rat nasal mucosa, J. Appl. Physiol 74 (1993) (1985) 2456–2461. [DOI] [PubMed] [Google Scholar]
[64].Diekmann O, Tschesche H, Degradation of kinins, angiotensins and substance P by polymorphonuclear matrix metalloproteinases MMP 8 and MMP 9, Braz. J. Med. Biol. Res 27 (1994) 1865–1876. [PubMed] [Google Scholar]
[65].Demattos RB, Bales KR, Cummins DJ, Dodart JC, Paul SM, Holtzman DM, Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases brain A beta burden in a mouse model of Alzheimer's disease, Proc. Natl. Acad. Sci. U. S. A 98 (2001) 8850–8855. [DOI] [PMC free article] [PubMed] [Google Scholar]
[66].Engelhardt B, Carare RO, Bechmann I, Flügel A, Laman JD, Weller RO, Vascular, glial, and lymphatic immune gateways of the central nervous system, Acta Neuropathol. 132 (2016) 317–338. [DOI] [PMC free article] [PubMed] [Google Scholar]
[67].Louveau A, Harris TH, Kipnis J, Revisiting the mechanisms of CNS immune privilege, Trends Immunol. 36 (2015) 569–577. [DOI] [PMC free article] [PubMed] [Google Scholar]
[68].McLendon R, Enterline D, Tien R, Thorstad W, Bruner J, Tumors of central neuroepithelial origin, in: Bigner D, McLendon R, Bruner J. (Eds.), Russell and Rubin stein's Pathology of Tumors of the Nervous System, CRC Press, London, 1998, pp. 523–528. [Google Scholar]
[69].Rickert CH, Extraneural metastases of paediatric brain tumours, Acta Neuropathol. 105 (2003) 309–327. [DOI] [PubMed] [Google Scholar]
[70].Bradbury MWB, Drainage of cerebral interstitial fluid and of cerebrospinal fluid into lymphatics, in: Cserr HF, Johnston MG (Eds.), Experimental Biology of the Lymphatic Circulation, Elsevier, New York and Amsterdam, 1985, pp. 355–391. [Google Scholar]
[71].Kida S, Pantazis A, Weller RO, CSF drains directly from the subarachnoid space into nasal lymphatics in the rat. Anatomy, histology and immunological significance, Neuropathol. Appl. Neurobiol 19 (1993) 480–488. [DOI] [PubMed] [Google Scholar]
[72].Bradbury MW, Westrop RJ, Factors influencing exit of substances from cerebrospinal fluid into deep cervical lymph of the rabbit, J. Physiol 339 (1983) 519–534. [DOI] [PMC free article] [PubMed] [Google Scholar]
[73].Cserr HF, Harling-Berg CJ, Knopf PM, Drainage of brain extracellular fluid into blood and deep cervical lymph and its immunological significance, Brain Pathol. 2 (1992) 269–276. [DOI] [PubMed] [Google Scholar]
[74].Faber W, The nasal mucosa and the subarachnoid space, Am. J. Anat 62 (1937) 121–148. [Google Scholar]
[75].Jansson B, Björk E, Visualization of in vivo olfactory uptake and transfer using fluorescein dextran, J. Drug Target 10 (2002) 379–386. [DOI] [PubMed] [Google Scholar]
[76].Sakane T, Akizuki M, Taki Y, Yamashita S, Sezaki H, Nadai T, Direct drug transport from the rat nasal cavity to the cerebrospinal fluid: the relation to the molecular weight of drugs, J. Pharm. Pharmacol 47 (1995) 379–381. [DOI] [PubMed] [Google Scholar]
[77].de Lange EC, Danhof M, Considerations in the use of cerebrospinal fluid pharmacokinetics to predict brain target concentrations in the clinical setting: implications of the barriers between blood and brain, Clin. Pharmacokinet 41 (2002) 691–703. [DOI] [PubMed] [Google Scholar]
[78].Veening JG, de Jong T, Barendregt HP, Oxytocin-messages via the cerebrospinal fluid: behavioral effects; a review, Physiol. Behav 101 (2010) 193–210. [DOI] [PubMed] [Google Scholar]
[79].Veening JG, Olivier B, Intranasal administration of oxytocin: behavioral and clinical effects, a review, Neurosci. Biobehav. Rev 37 (2013) 1445–1465. [DOI] [PMC free article] [PubMed] [Google Scholar]
[80].Dal Monte O, Noble PL, Turchi J, Cummins A, Averbeck BB, CSF and blood oxytocin concentration changes following intranasal delivery in macaque, PLoS One 9 (2014) e103677. [DOI] [PMC free article] [PubMed] [Google Scholar]
[81].Striepens N, Kendrick KM, Hanking V, Landgraf R, Wüllner U, Maier W, Hurlemann R, Elevated cerebrospinal fluid and blood concentrations of oxytocin following its intranasal administration in humans, Sci. Rep 3 (2013) 3440. [DOI] [PMC free article] [PubMed] [Google Scholar]
[82].Brambell FW, The transmission of immune globulins from the mother to the foetal and newborn young, Proc. Nutr. Soc 28 (1969) 35–41. [PubMed] [Google Scholar]
[83].Hutchings M, Weller RO, Anatomical relationships of the pia mater to cerebral blood vessels in man, J. Neurosurg 65 (1986) 316–325. [DOI] [PubMed] [Google Scholar]
[84].Federoff S, Juurlink BHJ, Doucette R, Biology and Pathology of Astrocyte-Neuron Interactions, Springer Science + Business Media LLC, 1993. [Google Scholar]

Associated Data

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Supplementary Materials

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NIHMS1504903-supplement-2.eps^{(1.1MB, eps)}

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[R1] [1].Yu CY, Ng G, Liao P, Therapeutic antibodies in stroke, Transl. Stroke Res 4 (2013) 477–483. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R2] [2].Lannfelt L, Möller C, Basun H, Osswald G, Sehlin D, Satlin A, Logovinsky V, Gellerfors P, Perspectives on future Alzheimer therapies: amyloid-β protofibrils - a new target for immunotherapy with BAN2401 in Alzheimer's disease, Alzheimers Res. Ther 6 (2014) 16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R3] [3].George S, Brundin P, Immunotherapy in Parkinson's disease: micromanaging alpha-Synuclein aggregation, J. Parkinsons Dis 5 (2015) 413–424. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] [4].Chacko AM, Li C, Pryma DA, Brem S, Coukos G, Muzykantov V, Targeted delivery of antibody-based therapeutic and imaging agents to CNS tumors: crossing the blood-brain barrier divide, Expert Opin. Drug Deliv 10 (2013) 907–926. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] [5].Rommer PS, Dudesek A, Stüve O, Zettl UK, Monoclonal antibodies in treatment of multiple sclerosis, Clin. Exp. Immunol 175 (2014) 373–384. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] [6].Hammarlund-Udenaes M, de Lange E, Thorne R, Drug Delivery to the Brain, 1 ed., AAPSPress. Springer, 2014. [Google Scholar]

[R7] [7].Reese TS, Karnovsky MJ, Fine structural localization of a blood-brain barrier to exogenous peroxidase, J. Cell Biol 34 (1967) 207–217. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R8] [8].Becker NH, Novikoff AB, Zimmerman HM, Fine structure observations of the uptake of intravenously injected peroxidase by the rat choroid plexus, J. Histochem. Cytochem 15 (1967) 160–165. [DOI] [PubMed] [Google Scholar]

[R9] [9].Nabeshima S, Reese TS, Landis DM, Brightman MW, Junctions in the meninges and marginal glia, J. Comp. Neurol 164 (1975) 127–169. [DOI] [PubMed] [Google Scholar]

[R10] [10].Poduslo JF, Curran GL, Berg CT, Macromolecular permeability across the blood-nerve and blood-brain barriers, Proc. Natl. Acad. Sci. U. S. A 91 (1994) 5705–5709. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] [11].Davson H, Segal M, Physiology of the CSF and Blood-brain Barriers, CRC Press, 1995. [Google Scholar]

[R12] [12].Balin BJ, Broadwell RD, Salcman M, El-Kalliny M, Avenues for entry of peripherally administered protein to the central nervous system in mouse, rat, and squirrel monkey, J. Comp. Neurol 251 (1986) 260–280. [DOI] [PubMed] [Google Scholar]

[R13] [13].Broadwell RD, Sofroniew MV, Serum proteins bypass the blood-brain fluid barriers for extracellular entry to the central nervous system, Exp. Neurol 120 (1993) 245–263. [DOI] [PubMed] [Google Scholar]

[R14] [14].Banks WA, Terrell B, Farr SA, Robinson SM, Nonaka N, Morley JE, Passage of amyloid beta protein antibody across the blood-brain barrier in a mouse model of Alzheimer's disease, Peptides 23 (2002) 2223–2226. [DOI] [PubMed] [Google Scholar]

[R15] [15].Wang Q, Delva L, Weinreb PH, Pepinsky RB, Graham D, Veizaj E, Cheung AE, Chen W, Nestorov I, Rohde E, Caputo R, Kuesters GM, Bohnert T, Gan LS, Monoclonal antibody exposure in rat and cynomolgus monkey cerebrospinal fluid following systemic administration, Fluids Barriers CNS 15 (2018) 10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] [16].Levites Y, Smithson LA, Price RW, Dakin RS, Yuan B, Sierks MR, Kim J, McGowan E, Reed DK, Rosenberry TL, Das P, Golde TE, Insights into the mechanisms of action of anti-Abeta antibodies in Alzheimer's disease mouse models, FASEB J. 20 (2006) 2576–2578. [DOI] [PubMed] [Google Scholar]

[R17] [17].St-Amour I, Paré I, Alata W, Coulombe K, Ringuette-Goulet C, Drouin-Ouellet J, Vandal M, Soulet D, Bazin R, Calon F, Brain bioavailability of human intravenous immunoglobulin and its transport through the murine blood-brain barrier, J. Cereb. Blood Flow Metab 33 (2013) 1983–1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] [18].Petereit HF, Rubbert-Roth A, Rituximab levels in cerebrospinal fluid of patients with neurological autoimmune disorders, Mult. Scler 15 (2009) 189–192. [DOI] [PubMed] [Google Scholar]

[R19] [19].Lochhead JJ, Thorne RG, Intranasal delivery of biologics to the central nervous system, Adv. Drug Deliv. Rev 64 (2012) 614–628. [DOI] [PubMed] [Google Scholar]

[R20] [20].Lochhead J, Thorne R, Intranasal drug delivery to the brain, in: Hammarlund-Udenaes D, Lange Thorne (Eds.), Drug Delivery to the Brain - Physiological concepts, Methodologies and Approaches, Springer, 2014, pp. 401–431. [Google Scholar]

[R21] [21].Dhuria SV, Hanson LR, Frey WH, Intranasal delivery to the central nervous system: mechanisms and experimental considerations, J. Pharm. Sci 99 (2010) 1654–1673. [DOI] [PubMed] [Google Scholar]

[R22] [22].Illum L, Nasal drug delivery - recent developments and future prospects, J. Control. Release 161 (2012) 254–263. [DOI] [PubMed] [Google Scholar]

[R23] [23].Thorne RG, Pronk GJ, Padmanabhan V, Frey WH, Delivery of insulin-like growth factor-I to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration, Neuroscience 127 (2004) 481–496. [DOI] [PubMed] [Google Scholar]

[R24] [24].Thorne RG, Hanson LR, Ross TM, Tung D, Frey WH, Delivery of interferon-beta to the monkey nervous system following intranasal administration, Neuroscience 152 (2008) 785–797. [DOI] [PubMed] [Google Scholar]

[R25] [25].Lochhead JJ, Wolak DJ, Pizzo ME, Thorne RG, Rapid transport within cerebral perivascular spaces underlies widespread tracer distribution in the brain after intranasal administration, J. Cereb. Blood Flow Metab 35 (2015) 371–381. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] [26].Asgari M, Zélicourt D, Kurtcuoglu V, Glymphatic solute transport does not require bulk flow, Sci Rep 6 (2016) 38635. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R27] [27].Kolobov VV, Davydova TV, Zakharova IA, Gorbatov VIU, Fomina VG, Repressional effects of the glutamate antibodies on expression of Dffb gene in the brain of rats with experimental Alzheimer's disease, Mol. Biol. (Mosk.) 46 (2012) 757–765. [PubMed] [Google Scholar]

[R28] [28].Kolobov VV, Zakharova IA, Fomina VG, Gorbatov VY, Davydova TV, Effect of antibodies to glutamate on caspase-3 activity in brain structures of rats with experimental Alzheimer's disease, Bull. Exp. Biol. Med 154 (2013) 425–427. [DOI] [PubMed] [Google Scholar]

[R29] [29].Chauhan MB, Chauhan NB, Brain uptake of neurotherapeutics after intranasal versus intraperitoneal delivery in mice, J. Neurol. Neurosurg 2 (2015). [PMC free article] [PubMed] [Google Scholar]

[R30] [30].Cattepoel S, Hanenberg M, Kulic L, Nitsch RM, Chronic intranasal treatment with an anti-Aβ(30–42) scFv antibody ameliorates amyloid pathology in a trans-genic mouse model of Alzheimer's disease, PLoS One 6 (2011) e18296. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] [31].Kumar NN, Gautam M, Lochhead JJ, Wolak DJ, Ithapu V, Singh V, Thorne RG, Relative vascular permeability and vascularity across different regions of the rat nasal mucosa: implications for nasal physiology and drug delivery, Sci. Rep 6 (2016) 31732. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R32] [32].Frey W, Delivery of 125I-NGF to the brain via the olfactory route, Drug Deliv. 4 (1997) 87–92. [Google Scholar]

[R33] [33].Guide for the care and use of Laboratory Animals, The National Academies Press, Washington, D.C., 2011.

[R34] [34].Pizzo ME, Wolak DJ, Kumar NN, Brunette E, Brunnquell CL, Hannocks MJ, Abbott NJ, Meyerand ME, Sorokin L, Stanimirovic DB, Thorne RG, Intrathecal antibody distribution in the rat brain: surface diffusion, perivascular transport, and osmotic enhancement of delivery, J. Physiol 596 (3) (2018) 445–475. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R35] [35].Schmitt C, Strazielle N, Ghersi-Egea JF, Brain leukocyte infiltration initiated by peripheral inflammation or experimental autoimmune encephalomyelitis occurs through pathways connected to the CSF-filled compartments of the forebrain and midbrain, J. Neuroinflammation 9 (2012) 187. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R36] [36].Fraker PJ, Speck JC, Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril, Biochem. Biophys. Res. Commun 80 (1978) 849–857. [DOI] [PubMed] [Google Scholar]

[R37] [37].Paxinos G, The Rat Nervous System, 4th ed., Elsevier, London, 2015. [Google Scholar]

[R38] [38].Thorne R, Primer on central nervous system structure/function and the vasculature, ventricular system, and fluids of the brain, in: Hammarlund-Udenaes D, Lange Thorne (Eds.), Drug Delivery to the Brain - Physiological Concepts, Methodologies and Approaches, Springer, 2014, pp. 685–707. [Google Scholar]

[R39] [39].Palkovits M, Brownstein MJ, Maps and Guide to Microdissection of the Rat Brain, Elsevier, New York, 1988. [Google Scholar]

[R40] [40].Paxinos G, Watson C, The Rat Brain in Stereotaxic Co-ordinates, 6th ed., Elsevier Inc., 2007. [Google Scholar]

[R41] [41].Bleys RL, Groen GJ, Hommersom RF, Neural connections in and around the cavernous sinus in rat, with special reference to cerebrovascular innervation, J. Comp. Neurol 369 (1996) 277–291. [DOI] [PubMed] [Google Scholar]

[R42] [42].Aspelund A, Antila S, Proulx ST, Karlsen TV, Karaman S, Detmar M, Wiig H, Alitalo K, A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules, J. Exp. Med 212 (2015) 991–999. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R43] [43].Abbott NJ, Pizzo ME, Preston JE, Janigro D, Thorne RG, The role of brain barriers in fluid movement in the CNS: is there a 'glymphatic' system? Acta Neuropathol. 135 (2018) 387–407. [DOI] [PubMed] [Google Scholar]

[R44] [44].Bien-Ly N, Boswell CA, Jeet S, Beach TG, Hoyte K, Luk W, Shihadeh V, Ulufatu S, Foreman O, Lu Y, Devoss J, van der Brug M, Watts RJ, Lack of widespread BBB disruption in Alzheimer's disease models: focus on therapeutic antibodies, Neuron 88 (2015) 289–297. [DOI] [PubMed] [Google Scholar]

[R45] [45].Deli MA, Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery, Biochim. Biophys. Acta 1788 (2009) 892–910. [DOI] [PubMed] [Google Scholar]

[R46] [46].Marttin E, Verhoef JC, Cullander C, Romeijn SG, Nagelkerke JF, Merkus FW, Confocal laser scanning microscopic visualization of the transport of dextrans after nasal administration to rats: effects of absorption enhancers, Pharm. Res 14 (1997) 631–637. [DOI] [PubMed] [Google Scholar]

[R47] [47].Yabluchanskiy A, Ma Y, Iyer RP, Hall ME, Lindsey ML, Matrix metalloprotei-nase-9: many shades of function in cardiovascular disease, Physiology (Bethesda) 28 (2013) 391–403. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R48] [48].Hackel D, Krug SM, Sauer RS, Mousa SA, Böcker A, Pflücke D, Wrede EJ, Kistner K, Hoffmann T, Niedermirtl B, Sommer C, Bloch L, Huber O, Blasig IE, Amasheh S, Reeh PW, Fromm M, Brack A, Rittner HL, Transient opening of the perineurial barrier for analgesic drug delivery, Proc. Natl. Acad. Sci. U. S. A 109 (2012) E2018–E2027. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R49] [49].Wolburg H, Wolburg-Buchholz K, Sam H, Horvát S, Deli MA, Mack AF, Epithelial and endothelial barriers in the olfactory region of the nasal cavity of the rat, Histochem. Cell Biol 130 (2008) 127–140. [DOI] [PubMed] [Google Scholar]

[R50] [50].Gueye Y, Ferhat L, Sbai O, Bianco J, Ould-Yahoui A, Bernard A, Charrat E, Chauvin JP, Risso JJ, Féron F, Rivera S, Khrestchatisky M, Trafficking and secretion of matrix metalloproteinase-2 in olfactory ensheathing glial cells: a role in cell migration? Glia 59 (2011) 750–770. [DOI] [PubMed] [Google Scholar]

[R51] [51].Li Y, Field PM, Raisman G, Olfactory ensheathing cells and olfactory nerve fibroblasts maintain continuous open channels for regrowth of olfactory nerve fibres, Glia 52 (2005) 245–251. [DOI] [PubMed] [Google Scholar]

[R52] [52].Wigley C, Nervous system, 40th ed., Churchill Livingstone Elsevier, Spain, 2008. [Google Scholar]

[R53] [53].Mery S, Gross EA, Joyner DR, Godo M, Morgan KT, Nasal diagrams: a tool for recording the distribution of nasal lesions in rats and mice, Toxicol. Pathol 22 (1994) 353–372. [DOI] [PubMed] [Google Scholar]

[R54] [54].Harkema JR, Carey SA, Wagner JG, The nose revisited: a brief review of the comparative structure, function, and toxicologic pathology of the nasal epithelium, Toxicol. Pathol 34 (2006) 252–269. [DOI] [PubMed] [Google Scholar]

[R55] [55].Adolfsson O, Pihlgren M, Toni N, Varisco Y, Buccarello AL, Antoniello K, Lohmann S, Piorkowska K, Gafner V, Atwal JK, Maloney J, Chen M, Gogineni A, Weimer RM, Mortensen DL, Friesenhahn M, Ho C, Paul R, Pfeifer A, Muhs A, Watts RJ, An effector-reduced anti-β-amyloid (Aβ) antibody with unique aβ binding properties promotes neuroprotection and glial engulfment of Aβ, J. Neurosci 32 (2012) 9677–9689. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R56] [56].Fuller JP, Stavenhagen JB, Christensen S, Kartberg F, Glennie MJ, Teeling JL, Comparing the efficacy and neuroinflammatory potential of three anti-abeta anti-bodies, Acta Neuropathol. 130 (2015) 699–711. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R57] [57].Arvidsson B, Kristensson K, Olsson Y, Vascular permeability to fluorescent protein tracer in trigeminal nerve and gasserian ganglion, Acta Neuropathol. 26 (1973) 199–205. [DOI] [PubMed] [Google Scholar]

[R58] [58].Matter K, Balda MS, SnapShot: epithelial tight junctions, Cell 157 (2014) 992. [DOI] [PubMed] [Google Scholar]

[R59] [59].Altner H, Altner-Kolnberger I, Freeze-fracture and tracer experiments on the permeability of the zonulae occludentes in the olfactory mucosa of vertebrates, Cell Tissue Res. 154 (1974) 51–59. [DOI] [PubMed] [Google Scholar]

[R60] [60].Ye L, Zeng R, Bai Y, Roopenian DC, Zhu X, Efficient mucosal vaccination mediated by the neonatal Fc receptor, Nat. Biotechnol 29 (2011) 158–163. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R61] [61].Heidl S, Ellinger I, Niederberger V, Waltl EE, Fuchs R, Localization of the human neonatal Fc receptor (FcRn) in human nasal epithelium, Protoplasma 253 (2016) 1557–1564. [DOI] [PubMed] [Google Scholar]

[R62] [62].Finger T, Silver W, The Neurobiology of Taste and Smell, 2 ed., (1987). [Google Scholar]

[R63] [63].Bertrand C, Geppetti P, Baker J, Petersson G, Piedimonte G, Nadel JA, Role of peptidases and NK1 receptors in vascular extravasation induced by bradykinin in rat nasal mucosa, J. Appl. Physiol 74 (1993) (1985) 2456–2461. [DOI] [PubMed] [Google Scholar]

[R64] [64].Diekmann O, Tschesche H, Degradation of kinins, angiotensins and substance P by polymorphonuclear matrix metalloproteinases MMP 8 and MMP 9, Braz. J. Med. Biol. Res 27 (1994) 1865–1876. [PubMed] [Google Scholar]

[R65] [65].Demattos RB, Bales KR, Cummins DJ, Dodart JC, Paul SM, Holtzman DM, Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases brain A beta burden in a mouse model of Alzheimer's disease, Proc. Natl. Acad. Sci. U. S. A 98 (2001) 8850–8855. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R66] [66].Engelhardt B, Carare RO, Bechmann I, Flügel A, Laman JD, Weller RO, Vascular, glial, and lymphatic immune gateways of the central nervous system, Acta Neuropathol. 132 (2016) 317–338. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R67] [67].Louveau A, Harris TH, Kipnis J, Revisiting the mechanisms of CNS immune privilege, Trends Immunol. 36 (2015) 569–577. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R68] [68].McLendon R, Enterline D, Tien R, Thorstad W, Bruner J, Tumors of central neuroepithelial origin, in: Bigner D, McLendon R, Bruner J. (Eds.), Russell and Rubin stein's Pathology of Tumors of the Nervous System, CRC Press, London, 1998, pp. 523–528. [Google Scholar]

[R69] [69].Rickert CH, Extraneural metastases of paediatric brain tumours, Acta Neuropathol. 105 (2003) 309–327. [DOI] [PubMed] [Google Scholar]

[R70] [70].Bradbury MWB, Drainage of cerebral interstitial fluid and of cerebrospinal fluid into lymphatics, in: Cserr HF, Johnston MG (Eds.), Experimental Biology of the Lymphatic Circulation, Elsevier, New York and Amsterdam, 1985, pp. 355–391. [Google Scholar]

[R71] [71].Kida S, Pantazis A, Weller RO, CSF drains directly from the subarachnoid space into nasal lymphatics in the rat. Anatomy, histology and immunological significance, Neuropathol. Appl. Neurobiol 19 (1993) 480–488. [DOI] [PubMed] [Google Scholar]

[R72] [72].Bradbury MW, Westrop RJ, Factors influencing exit of substances from cerebrospinal fluid into deep cervical lymph of the rabbit, J. Physiol 339 (1983) 519–534. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R73] [73].Cserr HF, Harling-Berg CJ, Knopf PM, Drainage of brain extracellular fluid into blood and deep cervical lymph and its immunological significance, Brain Pathol. 2 (1992) 269–276. [DOI] [PubMed] [Google Scholar]

[R74] [74].Faber W, The nasal mucosa and the subarachnoid space, Am. J. Anat 62 (1937) 121–148. [Google Scholar]

[R75] [75].Jansson B, Björk E, Visualization of in vivo olfactory uptake and transfer using fluorescein dextran, J. Drug Target 10 (2002) 379–386. [DOI] [PubMed] [Google Scholar]

[R76] [76].Sakane T, Akizuki M, Taki Y, Yamashita S, Sezaki H, Nadai T, Direct drug transport from the rat nasal cavity to the cerebrospinal fluid: the relation to the molecular weight of drugs, J. Pharm. Pharmacol 47 (1995) 379–381. [DOI] [PubMed] [Google Scholar]

[R77] [77].de Lange EC, Danhof M, Considerations in the use of cerebrospinal fluid pharmacokinetics to predict brain target concentrations in the clinical setting: implications of the barriers between blood and brain, Clin. Pharmacokinet 41 (2002) 691–703. [DOI] [PubMed] [Google Scholar]

[R78] [78].Veening JG, de Jong T, Barendregt HP, Oxytocin-messages via the cerebrospinal fluid: behavioral effects; a review, Physiol. Behav 101 (2010) 193–210. [DOI] [PubMed] [Google Scholar]

[R79] [79].Veening JG, Olivier B, Intranasal administration of oxytocin: behavioral and clinical effects, a review, Neurosci. Biobehav. Rev 37 (2013) 1445–1465. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R80] [80].Dal Monte O, Noble PL, Turchi J, Cummins A, Averbeck BB, CSF and blood oxytocin concentration changes following intranasal delivery in macaque, PLoS One 9 (2014) e103677. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R81] [81].Striepens N, Kendrick KM, Hanking V, Landgraf R, Wüllner U, Maier W, Hurlemann R, Elevated cerebrospinal fluid and blood concentrations of oxytocin following its intranasal administration in humans, Sci. Rep 3 (2013) 3440. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R82] [82].Brambell FW, The transmission of immune globulins from the mother to the foetal and newborn young, Proc. Nutr. Soc 28 (1969) 35–41. [PubMed] [Google Scholar]

[R83] [83].Hutchings M, Weller RO, Anatomical relationships of the pia mater to cerebral blood vessels in man, J. Neurosurg 65 (1986) 316–325. [DOI] [PubMed] [Google Scholar]

[R84] [84].Federoff S, Juurlink BHJ, Doucette R, Biology and Pathology of Astrocyte-Neuron Interactions, Springer Science + Business Media LLC, 1993. [Google Scholar]

PERMALINK

Delivery of immunoglobulin G antibodies to the rat nervous system following intranasal administration: Distribution, dose-response, and mechanisms of delivery

Niyanta N Kumar

Jeffrey J Lochhead

Michelle E Pizzo

Geetika Nehra

Sam Boroumand

Gretchen Greene

Robert G Thorne

Abstract

1. Introduction

2. Methods

2.1. Experimental design and statistical analysis

2.2. Animals and surgeries

2.3. Intranasally and intra-arterially administered molecules

2.3.1. Radiolabeled antibodies

2.3.2. Fluorescently labeled antibodies

Matrix metalloproteinase-9 (MMP-9):

2.4. Intranasal and intra-arterial administration paradigm

2.4.1. Intranasal dosing

2.4.2. Intra-arterial dosing

2.4.3. Euthanasia and perfusion-fixation of tissue

2.5. Methods to measure tissue [125I]-IgG antibody concentration and distribution in the blood, CSF, and CNS

2.5.1. Blood sampling

2.5.2. CSF sampling via intracisternal withdrawal

2.5.3. Microdissection of major ventral cerebral arteries

Fig. 1.

2.5.4. Brain and cervical spinal cord microdissection

2.5.5. Trigeminal nerve dissection

2.5.6. Radioactivity measurement

2.6. Investigating the intactness of intranasally administered [125I]-IgG in the brain

2.7. Fluorescence imaging to study AF488-IgG distribution in the nasal passages, trigeminal nerves, and brain

2.7.1. Ex vivo fluorescence microscopy

2.7.2. Immunofluorescence and confocal microscopy

3. Results

3.1. Intranasal and intra-arterial delivery of different doses of [125I]-IgG resulted in similar endpoint blood concentrations

Table 1.

Table 2.

Table 3.

3.2. A low 50 μg tracer [125I]-IgG dose accessed the CNS and bypassed the BBB and BCSFBs following intranasal administration

3.3. Two higher [125I]-IgG doses (1 mg and 2.5 mg) accessed the CNS and bypassed the BBB and BCSFBs following intranasal administration

3.4. [125I]-IgG heavy and light chains were detected in the brain soluble protein fraction following intranasal delivery

3.5. Dose-response: intranasal but not intra-arterial administration of higher [125I]-IgG doses resulted in significantly higher CNS IgG concentrations

Fig. 2.

3.6. Intranasal matrix metalloproteinase-9 pre-treatment enhanced nasally applied [125I]-IgG access to the nervous system

Table 4.

Fig. 3.

3.7. Intranasal administration resulted in a widespread autoradiographic distribution of [125I]-IgG within the CNS that corresponded with brain entry pathways

Fig. 4.

3.8. Imaging of fluorophore-labeled IgG distribution following intranasal administration revealed olfactory and trigeminal brain entry pathways and further distribution within the CNS via perivascular spaces

Fig. 5.

Fig. 7.

Fig. 6.

4. Discussion

5. Conclusions

Supplementary Material

Acknowledgements

Footnotes

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

2.5. Methods to measure tissue [¹²⁵I]-IgG antibody concentration and distribution in the blood, CSF, and CNS

2.6. Investigating the intactness of intranasally administered [¹²⁵I]-IgG in the brain

3.1. Intranasal and intra-arterial delivery of different doses of [¹²⁵I]-IgG resulted in similar endpoint blood concentrations

3.2. A low 50 μg tracer [¹²⁵I]-IgG dose accessed the CNS and bypassed the BBB and BCSFBs following intranasal administration

3.3. Two higher [¹²⁵I]-IgG doses (1 mg and 2.5 mg) accessed the CNS and bypassed the BBB and BCSFBs following intranasal administration

3.4. [¹²⁵I]-IgG heavy and light chains were detected in the brain soluble protein fraction following intranasal delivery

3.5. Dose-response: intranasal but not intra-arterial administration of higher [¹²⁵I]-IgG doses resulted in significantly higher CNS IgG concentrations

3.6. Intranasal matrix metalloproteinase-9 pre-treatment enhanced nasally applied [¹²⁵I]-IgG access to the nervous system

3.7. Intranasal administration resulted in a widespread autoradiographic distribution of [¹²⁵I]-IgG within the CNS that corresponded with brain entry pathways