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. 2025 Nov 5;103(11):e70091. doi: 10.1002/jnr.70091

Rolipram‐Loaded PgP Nanotherapeutics via Intrathecal Administration Reduces Secondary Injury in a Rat Acute Moderate Contusion SCI Model

Zhen Liao 1, Jun Gao 1, Min Kyung Khang 1, Ken Webb 2, Megan Ryan Detloff 3, Jeoung Soo Lee 1,✉
PMCID: PMC12588807  PMID: 41192818

ABSTRACT

Spinal cord injury (SCI) triggers complex secondary injury mechanisms, resulting in long‐term impacts on sensory and motor function. Rolipram, a phosphodiesterase‐4 inhibitor, has shown promise in preserving/restoring cyclic adenosine monophosphate (cAMP) to reduce secondary injury responses, but its clinical application is hindered by poor solubility and systemic side effects. To overcome these challenges, we developed rolipram‐loaded poly(lactide‐co‐glycolide)‐graft‐polyethylenimine (PgP) nanoparticles (Rm‐PgP) to enable localized and sustained drug delivery. In our previous findings, Rm‐PgP administered via intraspinal injection restored cAMP levels at the lesion site, and reduced secondary injury after moderate, contusive SCI. In this study, we investigated the effect of single and repeat administration of Rm‐PgP by the clinically relevant intrathecal route immediately after injury. We observed that the hydrophobic dye, DiR‐loaded PgP (DiR‐PgP) was retained in the CNS over 7 days post‐injury (DPI). In addition, we observed that both single and repeat Rm‐PgP treatment groups showed higher cAMP levels compared to those in the untreated SCI group and only the single treatment group showed a significant difference compared to the untreated SCI group. Lastly, we observed that cAMP restoration in both single and repeat Rm‐PgP treatment groups showed higher levels of activated cAMP‐response element‐binding protein (pCREB) relative to the untreated control. We also observed that both Rm‐PgP treatment groups showed reduced inflammatory response, reduced astrogliosis and apoptosis, and increased neuronal survival and spared tissue volume. These findings highlight the neuroprotective efficacy of Rm‐PgP by intrathecal administration in mitigating secondary injury during the critical early phase of recovery after SCI.

Keywords: apoptosis, astrogliosis, nanocarrier, neuroinflammation, neuronal survival, rolipram, spinal cord injury

The biodistribution of DiR‐PgP and the therapeutic efficacy of Rm‐PgP after intrathecal administration was investigated in a rat moderate thoracic contusion SCI model. Intrathecal administration of DiR‐PgP showed CNS‐specific retention without peripheral distribution. Intrathecal administration of Rm‐PgP enhanced tissue sparing, increased pCREB/CREB ratio, and reduced inflammation and apoptosis, demonstrating PgP's potential for sustained CNS drug delivery and neuroprotection after SCI.

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Summary.

  • Rolipram, a phosphodiesterase (PDE4) inhibitor, represents a promising therapeutic strategy for neurotrauma by preserving and restoring intracellular cAMP levels.

  • A polymeric micelle nanoparticle, poly (lactide‐co‐glycolide)‐graft‐polyethylenimine (PgP), for targeted rolipram delivery to the injured spinal cord.

  • DiR loaded‐PgP (DiR‐PgP) administered via intrathecal injection demonstrated prolonged CNS residence and no distribution to peripheral organs.

  • In a rat moderate contusion SCI model, both single and repeat intrathecal Rm‐PgP treatment restored/preserved cAMP levels and increased pCREB/CREB ratio, indicating activation of pro‐survival signaling pathways.

  • Furthermore, both single and repeat intrathecal Rm‐PgP treatment reduced inflammatory response, astrogliosis, and apoptosis, while enhancing neuronal survival and tissue sparing at the injury site.

  • These findings highlight intrathecal Rm‐PgP treatment as a promising approach for sustained local rolipram delivery and functional neuroprotection following spinal cord injury.

1. Introduction

Spinal cord injury (SCI) disrupts neurological pathways and adversely impacts motor, sensory, respiratory, and bladder/bowel function depending upon the severity and location of the injury (Hagen 2015). Multiple mechanisms hinder recovery from SCI, including (1) a cascade of secondary injury processes causing progressive neurodegeneration, (2) injury‐induced changes in the neural microenvironment and (3) age‐related changes in intrinsic axonal growth capacity (Hachem and Fehlings 2021; Filous and Schwab 2018; Zheng and Tuszynski 2023). Therapeutic strategies targeting these barriers have been developed to provide neuroprotection and promote axonal regeneration. While promising results have been achieved in preclinical injury models, improvements reported in clinical trials have been modest (Badhiwala et al. 2019; Dalamagkas et al. 2018). Current clinical management focuses on early surgical decompression, managing neurological sequelae and secondary complications, and long‐term rehabilitation (Ahuja et al. 2017). Systemic methylprednisolone administered within 8 h post‐injury is the only approved treatment and remains controversial (Gupta et al. 2010). Thus, novel therapeutic approaches for SCI are urgently required.

One important acute consequence of SCI is a significant reduction in cyclic adenosine monophosphate (cAMP) levels in the spinal cord (Zhou et al. 2022), likely due to increased phosphodiesterase (PDE) activity (Houslay et al. 2005). cAMP is a pivotal second messenger and intracellular metabolite that signals through protein kinase A (PKA)/cAMP response element‐binding protein (CREB) and exchange protein activated by cyclic‐AMP 2 (EPAC2) (Neumann et al. 2002; Qiu et al. 2002). cAMP signaling plays a critical role in neurons, glia, and immune cells; supporting neuroprotection and repair by regulating peripheral immune cell recruitment and activation, inflammatory cytokine expression, astrocyte neurotrophin production, neuronal survival, and oligodendrocyte precursor cell differentiation (Zhou et al. 2022, 2019; Knott et al. 2017; Syed et al. 2013; Renz et al. 1988; Negreiros‐Lima et al. 2020). Consequently, cAMP has been a major target of therapeutic development for neurotrauma and neurodegenerative diseases (Mussen et al. 2023).

Several preclinical studies have investigated the administration of cAMP analogues, such as dibutyryl cAMP (Fouad et al. 2009; Kim et al. 2011) or PDE4 inhibitors such as rolipram and roflumilast (Bao et al. 2011; Moradi et al. 2020), to preserve/restore cAMP levels after SCI. Rolipram has been most widely studied because of its ability to cross the blood–brain barrier and has been shown in preclinical SCI models to provide neuroprotection, increase white matter sparing/oligodendrocyte survival, and improve motor functional recovery (Whitaker et al. 2008; Costa et al. 2013; Schaal et al. 2012; Macks et al. 2018; Gao et al. 2023). Rolipram has also been included in several combination therapies shown to improve recovery after SCI (Nikulina et al. 2004; Flora et al. 2013). Despite these encouraging results, clinical translation of rolipram has been hindered by delivery challenges including limited aqueous solubility necessitating initial dissolution in organic solvents such as alcohol or dimethyl sulfoxide (DMSO) and adverse side effects when administered systemically (Galvao et al. 2014). Therefore, there is a clear need for improved therapeutic strategies to safely deliver rolipram to the injured spinal cord.

Nanotechnology has emerged as a promising approach for drug delivery to the CNS, seeking to mitigate the side effects commonly associated with high‐dose systemic administration (Tyler et al. 2013; Mitragotri et al. 2014). Nanotechnology‐based drug delivery systems offer the potential for targeted and sustained delivery of therapeutic agents to the SCI injury site (Song et al. 2019; Park et al. 2019; Jeong et al. 2017). Furthermore, nanoparticles can protect therapeutic agents from degradation and increase their bioavailability, leading to improved therapeutic outcomes (Mitragotri et al. 2014). In addition to drug delivery, nanotechnology‐based scaffolds and matrices can also be used to support tissue engineering and regeneration of the spinal cord (Song et al. 2019). The application of nanotechnology in SCI holds great promise for developing more effective treatments to reduce the extent of secondary injury and promote neural repair and regeneration.

The goal of our research is to develop a nanocarrier that can increase the uptake and bioavailability of rolipram at the SCI injury site, thereby reducing secondary injury and promoting neural repair and regeneration. To achieve this goal, we developed an amphiphilic polymeric micellar delivery system, poly(lactide‐co‐glycolide)‐graft‐polyethylenimine (PgP). Previously, we showed that rolipram (Rm) can be loaded within the PgP hydrophobic core (Rm‐PgP), increasing its aqueous solubility 6–8‐fold and eliminating the need for organic solvents (Macks et al. 2018). Local delivery of Rm‐PgP by intraspinal injection at the SCI lesion epicenter restored cAMP levels, reduced secondary injury, and improved motor function (Macks et al. 2018; Gao et al. 2023). Here, we investigated for the first time the administration of Rm‐PgP by intrathecal catheter in a rat T9 mid‐thoracic moderate contusion SCI. We first evaluated PgP biodistribution and CNS residence time using a fluorescent dye. Then we examined the therapeutic efficacy of single and repeat Rm‐PgP administration on cAMP levels, CREB activation, apoptosis, astrogliosis, and inflammation at 7 days post injury (DPI).

2. Materials and Methods

2.1. Materials

Rolipram (Rm) was purchased from LC Laboratories (Woburn, MA). 1,1′‐Dioctadecyl‐3,3,3′,3′‐Tetramethylindotricarbocyanine Iodide (DiR) was obtained from Invitrogen (Eugene, OR). Acetone and ethanol were purchased from Sigma Aldrich (St. Louis, MO). Parameter mouse/rat cAMP Assay kit was purchased from R&D Systems (Minneapolis, MN). The ApopTag Fluorescein In Situ Apoptosis Detection Kit was obtained from EMD Millipore (Darmstadt, Germany).

2.2. Preparation of DiR‐PgP

PgP was synthesized and characterized as previously reported (Gwak et al. 2016). Briefly, PgP was synthesized using PLGA (4 kDa, 50:50, Durect Corporation, Cupertino, CA) containing carboxylic end groups and branched PEI (bPEI, 25 kDa, Sigma‐Aldrich, St. Louis, MO). DiR‐PgP was prepared as previously described (Macks et al. 2018) Briefly, DiR was first dissolved in acetone at a concentration of 2.5 mg/mL and PgP dissolved at 1 mg/mL in water. Then, 100 μL (250 μg of DiR) DiR solution was added into 1 mL PgP solution. The mixture was incubated 4 h while stirring in the dark and then the lid was opened to allow acetone to be evaporated overnight while stirring. The DiR‐PgP and DiR‐water was then filtered through a syringe filter (Molecular weight cut off (MWCO) = 0.2 μm, cellulose acetate, Nalgen, Thermoscientific) to remove precipitated DiR.

2.3. Preparation and Loading Efficiency of Rm‐Loaded PgP

Rm was loaded into PgP by the solvent evaporation as described in our published paper (Macks et al. 2018). Briefly, Rm was dissolved in ethanol (10 mg/mL) and PgP was dissolved in water (1 mg/mL). 100 μL (1 mg) of Rm solution was slowly added into 1 mL PgP solution. The tube was sealed and the mixture was gently stirred for 4 h at 350 rpm at room temperature. Then ethanol was evaporated overnight, and the solution was filtered (0.2 μm nylon syringe filter, Nalgene, Waltham, MA) to remove unloaded/precipitated Rm. The amount of Rm loaded in PgP was measured by High Performance Liquid Chromatography (HPLC; Waters, Milford, MA) composed of Waters 1525 binary HPLC pump, Waters 2998 photodiode array detector, and a Shodex C18 column (Showa Denko America, New York, NY). The mobile phase was water: acetonitrile (60:40). The injection volume was 20 mL and the run time was 6 min. The flow rate was 0.2 mL/min, and Rm was detected at 280 nm UV wavelength. The Rm loading efficiency was calculated as:

%Loading efficiency=Amount ofRmloaded/Amount ofRmadded×100

2.4. Generation of Rat Moderate Contusion SCI Model and Intrathecal Catheterization

All surgical procedures and post‐operative care were conducted under the supervision of the Clemson University Institutional Animal Care and Use Committee (approved animal protocol no. AUP 2019‐043). Sprague Dawley rats (male, 200–250 g, 7–8 weeks) were deeply anesthetized with ketamine/xylazine (80–100 mg/kg). Their backs were shaved and prepared with betadine solution, chlorohexidine, and sterile water. A 4‐cm longitudinal incision over the dorsal mid‐thoracic region was made and the T8–T10 spinous processes were identified. The lamina was removed by bone rongeur and the dura exposed. Next, the lumbar region of the spinal cord was exposed by dorsal laminectomy. After a small hole was made in the dura overlying L4–L5 using a 30G needle, a 32G catheter (ReCathCo LLC, Allison Park, PA, USA) was inserted under the dura and secured via suturing to both the dura and the neighboring muscle. The rat was then positioned and stabilized in the Infinite Horizon's spinal cord impactor (IH‐0400, PSI, Lexington, KY) and the 2.5 mm probe centered over the midline at T9. A contusion injury was generated with a force of 200 kDyne, speed of 120 mm/s, and 1 s dwell time.

Saline, dye‐loaded PgP (DiR‐PgP), and Rm‐PgP were administered via intrathecal catheter using a microinjection pump. Following intrathecal injection, the paraspinal muscles were closed with 4–0 vicryl suture (V310, Pivetal, Loveland, CO) and the skin was closed with a 9 mm surgical clip (Autoclip Wound Clips, Alimed, Dedham, MA). The catheter tube end was sealed with glue and then placed in a subcutaneous pocket. Cefazolin (50 mg/kg) and buprenorphine (0.25 mg/kg) were injected intraperitoneally, and 5 mL lactate solution was injected subcutaneously. Animals were kept warm until recovery from anesthesia. Bladders were expressed manually three times a day until normal bladder function returned.

2.5. Biodistribution of DiR‐PgP After Intrathecal Injection Immediately After Injury

A lipophilic, near‐infrared fluorescent cyanine dye, DiR, was used to visualize the biodistribution of PgP in a rat contusion SCI model after intrathecal injection. DiR‐PgP was administered immediately after injury via intrathecal injection using a microinjection pump system (UMP‐3 T, World Precision Instruments, Sarasota, FL) via intrathecal catheter (20 μL, 2 μL/min).

As shown in Scheme 1, the biodistribution of DiR‐PgP was imaged by the IVIS Live Animal Imaging System (IVIS, PerkinElmer, Waltham, MA) at 2 h, 1, 2, 3, 4, 5, 6 and 7 day(s) post‐intrathecal injection. At 1 day and 7 days post injury (DPI), animals were sacrificed by CO2 overdose and organs (brain, spinal cord, heart, liver, spleen, lung, and kidney) were harvested for ex vivo imaging by the IVIS system. Normal rats were used as a control to remove tissue autofluorescence background.

SCHEME 1.

SCHEME 1

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Blood distribution of DiR‐PgP after IT injection in a rat moderate contusion SCI model.

2.6. Effect of Single and Repeated Rm‐PgP Treatment on cAMP Level in Spinal Cord

Scheme 2 shows experimental design for cAMP level and histological analysis after single and repeat treatment of Rm‐PgP via intrathecal catheter immediately after SCI. Sixteen (n = 4/group) rats were used to assess cAMP levels in the spinal cord, and 20 rats (rat number/group: N = 2 for sham, n = 4 for SCI, n = 7 for Rm‐PgP‐S [single], and n = 7 Rm‐PgP‐R [repeat]) were used for histological analysis. Animals were divided into 4 groups: (1) Sham, (2) untreated SCI: 40 μL saline, (3) Rm‐PgP‐S: 40 μL Rm‐PgP (20 μg Rm) immediately after injury (Time 0), and (4) Rm‐PgP‐R: 40 μL Rm‐PgP (20 μg Rm) at 0, 2, and 4 DPI (Total 60 μg Rm).

SCHEME 2.

SCHEME 2

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Effect of Rm‐PgP after IT injection on cAMP level and secondary injury in a rat moderate contusion SCI model.

To evaluate the effect of Rm‐PgP treatment on cAMP level in lesion site, rats were deeply anesthetized with isoflurane gas (4% isoflurane in O2) and decapitated at 7 DPI as shown in Scheme 2. Spinal cords from the injury site (0.5 cm) were harvested and the cAMP level was measured by competitive ELISA using Parameter mouse/rat cAMP Assay kit (R&D Systems, Minneapolis, MN). Briefly, the spinal cords were rinsed with cold PBS and immediately placed in cold 0.1 N HCl. Spinal cords were homogenized with tissue homogenizer (TH‐01, OMNI international, America, GA), then the pH neutralized with 1 N NaOH at a 1:10 (v/v) of total sample volume. Samples were centrifuged at 600 × g for 10 min at 4°C to remove insoluble cellular debris and assayed immediately. 50 μL of mouse/rat cAMP conjugate and 100 μL sample or serially diluted cAMP standard were added to the biotinylated cAMP monoclonal antibody plate and incubated for 2 h at room temperature on the shaker. After stop solution addition, the optical density was measured at 450 and 570 nm wavelengths within 30 min using a microplate reader (Epoch, BioTek, America, CA). The concentration of cAMP in the samples was calculated based on the standard curve generated using Rm.

2.7. Histological Analysis of the Effect of Rm‐PgP

To evaluate the effect of Rm‐PgP treatment on secondary injury, rats (n = 2 for sham, n = 4 for SCI, n = 7 for Rm‐PgP‐S, and n = 7 for Rm‐PgP‐R) were deeply anesthetized with Euthasol (150 mg/kg) and sacrificed via cardiac perfusion using saline followed by 4% PFA at 7 DPI as shown in Scheme 2.

2.7.1. Tissue Collection and Sectioning

Spinal cord segments (1 cm long: 0.5 cm long rostral and caudal from epicenter) were isolated, post‐fixed in 4% paraformaldehyde solution for 48 h at 4°C followed by sucrose solution gradient (10%, 20%, and 30% sucrose) for 24 h in each solution at 4°C. The fixed tissue was then embedded in optical cutting temperature (OCT) compound (Fisher HealthCare, Houston, TX) at −20°C. The spinal cords were sectioned transversely (20 μm thickness) and mounted on positively charged glass slides in 10 series (each section was separated by 60 μm) and stored at −20°C.

2.7.2. Nissl Staining for Lesion Area

Sections (17 Sections/rat) were first placed in 2% w/v cresyl violet for 5 min, rinsed with distilled water, and then dehydrated for 5 min with 95% ethanol, 100% anhydrous ethanol and xylene. The stained sections were imaged by an all‐in‐one inverted fluorescence microscope (Keyence BZ‐X810, Keyence, Itasca, IL) and the lesion area was measured with NIH ImageJ 1.37v analysis software. Sham animals were used to normalize the spared tissue area. The spared tissue area of each section was calculated by subtracting the lesioned area from the cross‐sectional area of the section. The spared volume of the spinal cord was calculated by summing the spared tissue area of each section and then multiplying that by the distance between sections (in this instance, 0.6 mm).

The percentage of spared tissue volume was calculated by the equation below:

%spared tissue volume=Spared tissue volumeTotal tissue volume of sham*100%

2.7.3. Immunohistochemical Staining and Imaging

For immunohistochemistry, sections (Total 7 sections/rat, 1.2, 2.4 and 3.6 mm distances from the epicenter on both rostral (+) and caudal (−) sides) were selected and stained. For CREB and pCREB, sections were stained overnight at 4°C with antibodies for CREB (1:200; mouse anti‐CREB, Cat.# ab178322, Abcam, Cambridge, MA) and pCREB (phospho S133) (1:200; rabbit anti‐pCREB Cat.# ab32096, Abcam). For neuronal survival and astrogliosis, sections were stained overnight at 4°C with mouse monoclonal anti‐NeuN antibodies (1:200; Cat.# MAB377, Millipore) for neuronal nuclei and rabbit polyclonal anti‐GFAP (1:200; Cat.# ab7260, Abcam, Cambridge, MA) for activated astrocytes. For inflammatory response, sections were stained overnight at 4°C with rabbit monoclonal anti‐CD68 (1:200; Cat.# ab53004, Abcam, Cambridge, MA) for ED1+ cells and mouse monoclonal anti‐Arg1 antibodies (1:200; Cat.# C2720, Santa Cruz Biotechnology) for Arg1+ cells.

After incubation with primary antibodies as described above, sections were rinsed with PBST (1X PBS + 0.05% TritonX‐100), and then incubated with secondary antibodies for 2 h at room temperature. Goat anti‐mouse Cy3‐conjugated secondary antibodies (1:200; Cat.# 115–165‐003, Jackson ImmunoResearch, West Grove, PA) were used for CREB, NeuN and Arg1 and AlexaFluor 488‐conjugated goat anti‐rabbit secondary antibodies (1:500; Cat.# A‐11008, Thermo Fisher Scientific, Hampton, NH) were used for pCREB, GFAP and CD68.

For mounting, imaging, and analysis, anti‐fade mounting media with DAPI (Vectashield, Vector, Newark, CA) was applied and coverslips placed and sealed with clear nail polish (OPI, Calabasas, CA). Images of pCREB, CREB, NeuN, CD68, Arg1 and GFAP stained sections were captured using a Keyence BZ‐X810 microscope (Original magnification: 100X) and stitched by BZ‐X800 Analyzer. Positively stained cells were counted for all stains except GFAP using NIH imageJ1.37v analysis software.

GFAP staining was analyzed in 5 sections spanning the rostrocaudal extent of the injury site. Images were processed in ImageJ1.37v into 8‐bit images, and GFAP+ staining on each section was measured using the integrated density function. Integrated density is defined as the pixel intensity values within a selected region which is equivalent to the area of the selection multiplied by the mean gray value of the pixels. Data are collected in arbitrary units (AU) of fluorescence intensity/mm2. The integrated density of GFAP staining was determined for the sum of 5 sections throughout the rostrocaudal extent of the lesion.

2.7.4. Apoptosis by TUNEL Staining

Sections (Total 7 sections/rat, 1.2, 2.4, and 3.6 mm distances from the epicenter on both rostral (+) and caudal (−) sides) were selected and were stained by the terminal deoxynucleotidyl transferase‐mediated dUTP nicked end labeling (TUNEL) method using an ApopTag Plus Fluorescein in situ Apoptosis Detection Kit (S7111, EMD Millipore, Temecula, CA, USA) and anti‐fade mounting media with DAPI (Vectashield, Vector, Newark, CA) was applied and coverslips placed and sealed with clear nail polish (OPI, Calabasas, CA). Stained sections were imaged at 100X using a Keyence fluorescence microscope and TUNEL positive cells counted using NIH image software as described above. Sham animals were used as a control.

2.8. Statistical Analysis

All data are presented as the mean ± standard deviation. Statistical analysis was performed with one‐way ANOVA followed by a Dunnett multiple comparisons test (GraphPad Prism 10 software, CA, USA). Spared tissue area across the rostrocaudal extent of the lesion was analyzed via 2‐way repeated measures ANOVA (distance from the epicenter*treatment group). The results were considered statistically significant when a p value < 0.05 was obtained on a 2‐tailed test.

3. Results

3.1. Rm Loading Efficiency in PgP

The amount of rolipram (Rm) loaded in the PgP micelle was measured by HPLC as 1.08 ± 0.04 mg/mL (mean ± standard deviation, n = 5). This is 4.4 times higher than Rm's solubility in water (0.2 mg/mL). The consistency observed in loading efficiency across the replicates (n = 5) underscores the reproducibility of the loading process.

3.2. Biodistribution of DiR‐Loaded PgP After Intrathecal Injection

We observed that DiR‐PgP accumulated at the injury site of the spinal cord after intrathecal injection (Figure 1A). Figure 1B shows that DiR‐PgP was primarily retained in the injured spinal cord with some distribution to the brain over the course of 7 days. Normal, uninjured rats that did not receive DiR‐loaded PgP were used as a control to avoid autofluorescence by tissue. At 1 and 7 DPI, animals were sacrificed, and organs (spinal cord, brain, heart, lung, liver, spleen, and kidney) were isolated and imaged. We observed that most DiR‐PgP was retained in the spinal cords with strong fluorescence signals and in the brains with weak signals for up to 7 days, while no fluorescence signal was detected in other peripheral organs such as heart, lung, liver, spleen, and kidney (Figure 1C). These results demonstrate that DiR‐PgP did not distribute systemically and was retained in the CNS after intrathecal injection.

FIGURE 1.

FIGURE 1

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Biodistribution of DiR‐PgP administered by IT injection immediately after moderate T9 mid‐thoracic contusion SCI. (A) DiR‐PgP injection via intrathecal catheter and accumulation of DiR‐PgP in the spinal cord immediately after injection, (B) Images of live animals to visualize the distribution of DiR‐PgP at 2 h and up to 7 DPI (Left: Control). (C) Ex vivo Images of organs isolated to visualize the distribution of DiR‐PgP at 1 and 7 DPI (Left: Control).

3.3. Effect of Rm‐PgP Single and Repeated Treatment on cAMP Level in Spinal Cord

The effect of Rm‐PgP single and repeated treatment on cAMP preservation/restoration was evaluated at 7 DPI. The rats were sacrificed, spinal cord segments harvested, homogenized, and cAMP levels measured by ELISA. The cAMP level in the untreated SCI group was significantly lower than that in the sham group, while the cAMP level in both the single and repeated treatment groups was not significantly different compared to that in the sham group (Figure 2). Both Rm‐PgP single and repeated treatment groups had higher cAMP levels than that in the untreated SCI group, but only the single treatment group showed a significant difference compared to the untreated SCI group. There was no significant difference observed between the Rm‐PgP single and repeated treatment groups.

FIGURE 2.

FIGURE 2

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Effect of single and repeated intrathecal administration of Rm‐PgP on spinal cord cAMP levels at 7 DPI in a rat moderate contusion SCI model (n = 4/group). Sham: Laminectomy only, SCI: Saline injection, Rm‐PgP‐S: 40 μL Rm‐PgP (20 μg Rm) single injection at time 0, and Rm‐PgP‐R: 40 μL Rm‐PgP (20 μg Rm) injection at 0, 2, and 4 DPI. *p < 0.05, **p < 0.01 compared to SCI group. Data presented as mean ± standard deviation.

3.4. Effect of Rm‐PgP Single and Repeated Treatment on Spared Tissue Volume

The effect of Rm‐PgP single and repeated treatment on tissue sparing along a 1 cm segment of the spinal cord centered at the epicenter was evaluated by Nissl staining at 7 DPI (Figure 3). We observed that both single and repeated treatment significantly increased the spared tissue area in sections rostral to the SCI epicenter compared to untreated SCI. Further, the spared tissue volume in both Rm‐PgP single and repeated treatment groups was significantly greater compared with that of the untreated SCI group.

FIGURE 3.

FIGURE 3

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Effect of Rm‐PgP single and repeated treatment on spared tissue volume. (A) Diagram of section position (−: Rostral, Epi: Epicenter, +: Caudal). (B) Representative images of Nissl‐stained transverse spinal cord sections with lesion outlined in red. (Scale bar = 1 mm). (C) Spared tissue area from 17 equidistant (0.6 mm) sections throughout the 1 cm spinal cord segment. Data presented mean ± standard deviation (total 17 sections/rat, rat number/group: n = 4 rats for untreated SCI, n = 7 for Rm‐PgP‐S, and n = 7 for Rm‐PgP‐R, *SCI vs. Rm‐PgP‐S, Rm‐PgP‐R p < 0.05). (D) Spared volume of 1 cm segment of spinal cord centered on the lesion (*p < 0.05). Statistics: (C) Two‐way repeated measures ANOVA (Distance from epicenter*Treatment group p = 0.0261, F(5, 80) = 2.7 Treatment group p = 0.033, F(3, 16) = 3.7; Distance from epicenter*Treatment group p = 0.0003, F(48, 256) = 2.01); Tukey's multiple comparison test. (D) One‐way ANOVA p = 0.0191, F(2, 15) = 5.2.

3.5. Effect of Rm‐PgP Single and Repeated Treatment on pCREB/CREB

The effect of Rm‐PgP single and repeated treatment on pCREB/CREB was evaluated by IHC at 7 DPI. The CREB activation ratio pCREB/CREB was calculated as: (Total number of positive pCREB cells/Total number of CREB cells) × 100%. We observed that the percentage of pCREB+/CREB+ cells in both Rm‐PgP single and repeated treatment groups was higher than that in the untreated SCI group and the repeated group showed statistical significance compared to the untreated SCI group (Figure 4A). There was no significant difference between single and repeated treatment groups. Figure 4B shows representative images of sections stained for pCREB and CREB from sham, untreated SCI, Rm‐PgP‐S, and Rm‐PgP‐R groups.

FIGURE 4.

FIGURE 4

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Effect of Rm‐PgP single and repeated treatment on CREB expression and phosphorylation. (A) The ratio of pCREB+/CREB+: Data presented as mean ± standard deviation (total 7 sections/rat, rat number/group: n = 4 rats for untreated SCI, n = 7 for Rm‐PgP‐S, and n = 7 for Rm‐PgP‐R). *p < 0.05 compared with SCI untreated group. (B) Representative images of stained sections. Scale bar represents 0.1 mm.

3.6. Effect of Rm‐PgP Single and Repeated Treatment on Inflammatory Response

For the inflammatory response, sections were stained using antibodies for ED1 and Arg 1. We observed that the number of ED1+ cells in both Rm‐PgP single and repeated treatment groups was significantly lower than in the untreated SCI group (Figure 5A), while the numbers of Arg1+ cells in both Rm‐PgP single and repeated treatment groups were significantly higher than in the untreated SCI group (Figure 5B). Figure 5C shows representative images of stained sections from sham, untreated SCI, Rm‐PgP‐S, and Rm‐PgP‐R groups.

FIGURE 5.

FIGURE 5

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Effect of Rm‐PgP single and repeated treatment on inflammatory response. (A) Total number of ED1+ cells/mm2, (B) Total number of Arg + cells/mm2. Data presented mean ± STD (n = 4 for untreated SCI, n = 7 for Rm‐PgP‐S, and n = 7 for Rm‐PgP‐R), Data presented as mean ± STD. *p < 0.05 and ***p < 0.001 compared to untreated SCI. (C) Representative images of ED1+ positive cells in green and Arg1+ cells in red. Merged image included DAPI‐stained nuclei are in blue (Scale bar: 1 mm).

3.7. Effect of Rm‐PgP Single and Repeated Treatment on Neuronal Cell Survival and Astrogliosis

For neuronal survival and astrogliosis, sections were stained with antibodies for NeuN for neuronal nuclei and GFAP for activated astrocytes. We observed that the number of NeuN+ cells in both the Rm‐PgP single and repeated treatment groups was higher than that in the untreated SCI group, but the differences were not significant (Figure 6A). The percentage of fluorescence intensity from GFAP+ cells in both Rm‐PgP single and repeated treatment groups was significantly lower than that in the untreated SCI group (Figure 6B). Figure 6C shows representative images of stained sections from sham, untreated SCI, Rm‐PgP‐S, and Rm‐PgP‐R groups.

FIGURE 6.

FIGURE 6

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Effect of single and repeated intrathecal injection of Rm‐PgP on NeuN and GFAP expression. (A) Total NeuN+ cells/mm2. (B) Integrated density of GFAP+ cells: Data presented as mean ± standard deviation (total 7 sections/rat, rat number/group: n = 4 rats for untreated SCI, n = 7 for Rm‐PgP‐S, and n = 7 for Rm‐PgP‐R). *p < 0.05, compared to untreated SCI. (C) Representative images of NeuN+ positive cells in green and GFAP+ cells in red. DAPI stained nuclei are in blue (Scale bar: 1 mm).

3.8. Effect of Rm‐PgP Single and Repeat Treatment on Apoptosis

The effect of Rm‐PgP on apoptosis was assessed by TUNEL assay. The total number of TUNEL+ cells in the spinal cord at 7 different positions was counted. We observed that the total number of TUNEL+ cells in both Rm‐PgP single and repeated treatment groups was significantly lower than in the untreated SCI group (Figure 7A). Figure 7B shows representative images of TUNEL+ cells (green) in the epicenter from sham, untreated SCI, Rm‐PgP‐S, and Rm‐PgP‐R groups.

FIGURE 7.

FIGURE 7

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Effect of Rm‐PgP single and repeat treatment on apoptosis. (A) Total number of TUNEL+ cells counted in transverse sections from 7 different rostral caudal positions centered at the lesion epicenter and separated by 1.2 mm. Data presented as mean ± standard deviation (total 7 sections/rat, rat number/group: n = 4 rats for untreated SCI, n = 7 for Rm‐PgP‐S, and n = 7 for Rm‐PgP‐R). *p < 0.05 compared to untreated SCI. (B) Representative images of TUNEL+ positive cells in green and DAPI stained nuclei is in blue (Scale bar: 1 mm).

4. Discussion

The complex pathophysiology of SCI severely limits spontaneous recovery and presents multiple obstacles to the development of effective therapeutic treatments. Cyclic AMP (cAMP) is a central regulator of biochemical signaling in multiple neural cell types and therefore impacts a wide range of CNS physiological processes (Zhou et al. 2022; Mussen et al. 2023). Diminished cAMP has been implicated in a wide range of inflammatory pathologies, both in peripheral tissue and the CNS (Nourian et al. 2023; Levy and Zhou 2015). cAMP levels are controlled by a family of adenylate cyclase enzymes responsible for cAMP production and a large family of phosphodiesterase (PDE) enzymes responsible for its degradation. PDE inhibitors have been recognized as a promising class of pharmaceuticals for many years, but their clinical realization has been substantially hindered by significant adverse side effects, including nausea, emesis, and behavioral changes (Mori et al. 2010; Zhu et al. 2001). One approach to overcoming these challenges has been the development of new generations of PDE inhibitors with improved selectivity and specificity for individual PDE isotypes (Lugnier 2006; Blokland et al. 2019).

Another approach is the development of drug delivery systems such as nanoparticles or biomaterial implants that can increase bioavailability at the therapeutic target site while minimizing systemic distribution and associated side effects. Towards this end, we have developed poly (lactide‐co‐glycolide)‐graft‐polyethylenimine (PgP) that can efficiently deliver siRNA and pDNA in vitro and in rat native spinal cord in vivo (Gwak et al. 2016, 2017). In our previous study, we have demonstrated that rolipram can be loaded within its hydrophobic core and the amount of Rm loaded into PgP was approximately 7‐fold higher than its normal aqueous solubility (Macks et al. 2018). Local delivery of Rm by PgP (Rm‐PgP) to the injury epicenter restored cAMP levels for up to 3 days post‐injury (DPI) and mitigated secondary injury as determined by reductions in both apoptosis and the inflammatory response in a rat severe compression injury model (Macks et al. 2018). We also reported that acute local delivery of Rm‐PgP by intraspinal injection can restore cAMP level at the lesion site, and restored cAMP can improve motor functional recovery and reduce neuropathic pain after moderate, contusive SCI (Gao et al. 2023). However, intraspinal administration is not a clinically relevant route in human SCI patients. Therefore, we explored the potential of intrathecal (IT) administration as an alternative route that is less invasive and more clinically relevant than intraspinal administration.

In this study, we investigated intrathecal administration of PgP‐based nanoparticles in a moderate contusion SCI model, first examining biodistribution and then the effect of rolipram delivery on secondary injury responses. In order to investigate nanoparticle biodistribution after intrathecal injection, we first loaded PgP with the hydrophobic fluorescent dye DiR. We observed that most DiR‐PgP was retained in the spinal cords and brains for up to 7 days without distributing to peripheral organs such as liver, lung, heart, kidney, and spleen. These observations are consistent with our previous study in which DiR‐PgP administered by direct intraspinal injection was retained up to 5 days near the injection site (Macks et al. 2018). The prolonged CNS residence time provided by PgP‐based nanotherapeutics offers the potential for sustained, local drug delivery. The precise mechanism responsible for biodistribution properties of PgP remains unclear and merits further investigation in future studies.

After confirming the retention of DiR‐PgP in the CNS after intrathecal administration, we compared the effect of Rm‐PgP single and repeat injections on secondary injury at 7 DPI (acute phase of injury). In the case of cAMP level, both single and repeat Rm‐PgP treatment groups mitigated the significant injury‐induced decline observed in untreated SCI animals and preserved/restored cAMP levels at levels not significantly different from the sham animal group. In our previous studies, we also observed that cAMP level was restored to sham levels by Rm‐PgP single treatment via intraspinal injection in a rat severe compression model (Macks et al. 2018) and moderate contusion model (Gao et al. 2023). PKA and EPAC2 are two downstream effectors of cAMP that play a pivotal role in neuroprotection, neurite outgrowth, and regeneration in the mammalian CNS (Zhou et al. 2022; Robichaux and Cheng 2018). Peace et al., reported that elevated cAMP levels in the growth cone can activate PKA and EPAC and enhance neurite outgrowth by inhibiting the Rho‐ROCK pathway (Peace and Shewan 2011). In another study, activation of PKA and EPAC by cAMP in the cell body led to CREB phosphorylation and increased expression of genes related to neuronal survival and neurite outgrowth (Batty et al. 2017). In our histological analysis, we observed increased CREB activation (pCREB/CREB ratio) in both single and repeat Rm‐PgP treatment groups, suggesting increased cAMP‐dependent signaling.

Infiltrated macrophages and resident CNS microglia play an important role in the neuroinflammatory processes during the secondary injury phase after CNS trauma such as SCI. Indeed microglia/macrophages are dynamic and plastic cells in the healthy and diseased CNS, displaying a multidimensional state at any given time that encompasses morphological, transcriptional and functional states (Paolicelli et al. 2022). Facilitating changes in their state and their function has become an important therapeutic target for SCI repair (Kong and Gao 2017). Moradi et al. (2020) reported that intraperitoneal injection of the PDE4 inhibitor, roflumilast (0.25, 0.5, and 1 mg/kg) significantly increased cAMP levels compared to the untreated SCI group and was associated with decreased CD86 and increased CD163. In our study, we observed that Rm‐PgP (20 μg Rm/rats) intrathecal injection can significantly decrease the number of ED1 positive cells and increase the number of Arg 1positive cells, indicating that local rolipram delivery can modulate immune cell infiltration and microglial activation. We also observed that the ED1 positive cell number in the Rm‐PgP treatment group was reduced compared to the untreated SCI group in a rat compression SCI model at 7 DPI (Sub acute injury phase) (Macks et al. 2018) and in a rat moderate compression SCI model at 4 WPI (Chronic injury phase) (Gao et al. 2023). Myers et al. (2019), also reported that inflammatory protein markers of activated astrocytes (GFAP), macrophage/microglia (CD11b/Iba1), and the proinflammatory mediator Cox2, were decreased in PDE4 knockout mice and the absence of PDE4 improved white matter sparing and recovery of hindlimb locomotion following spinal cord injury. One interesting report by Schepers et al. (2024), is that they evaluated the effect of inhibiting specific PDE4 subtypes (PDE4B and PDE4D) on inflammatory and regenerative processes following SCI and they found that the PDE4D inhibitor Gebr32a improved functional as well as histopathological outcomes after SCI even when treated at 2 DPI, while the PDE4B inhibitor A33 did not improve functional as well as histopathological outcomes after SCI. Ultimately, the neuroinflammatory aspect of the secondary injury response and resulting pro‐inflammatory cytokine expression, imbalances in ion homeostasis, and free radical production can lead to substantial cell death and tissue destruction. Zhou et al. described in their review that cAMP elevating agents such as rolipram, roflumilast, and cAMP analogues increase cAMP levels in both neurons and glial cells and increased cAMP attenuates the inflammatory response, inhibits glial scar formation, and reduces the apoptosis of neurons and oligodendrocytes (Gupta et al. 2010). We observed that both single and repeat Rm‐PgP treatment increased neuronal survival and reduced astrogliosis and apoptosis as we observed in our previous studies (Macks et al. 2018; Gao et al. 2023). Mitigation of these secondary injury processes likely contributed to the observed increase in spared tissue volume.

5. Conclusion

We demonstrated that DiR‐PgP nanotherapeutics administered by intrathecal injection, a minimally invasive and clinically relevant route, exhibit prolonged residence time in the CNS without distribution to peripheral organs. Both single and repeat Rm‐PgP intrathecal administration were able to restore/preserve cAMP levels and reduce inflammatory responses, astrogliosis, and apoptosis and increase neuronal survival and tissue sparing at 7 DPI. Importantly, anatomical changes following Rm‐PgP treatment do not necessarily indicate functional improvements as we did not conduct behavioral or electrophysiological assessments in the present experiments. Even with varying degrees of tissue sparing at the injury epicenter, motor and sensory function can plateau at similar recovery levels without treatment (Kloos et al. 2005; Fouad et al. 2021). Another limitation of this study is that only male rats were used. In our future study, we will evaluate the effect of Rm‐PgP single and repeat treatment via intrathecal injection on motor function recovery and neuropathic pain, and secondary injury using both male and female rat moderate contusion SCI models. The other limitation of this study is that Rm‐PgP was administered immediately after injury, while patients cannot be treated immediately after injury in a clinical setting. We will evaluate the effect of Rm‐PgP treatment at delayed time points (1 [acute], 7 [sub‐acute], and 28 [chronic] DPI) via intrathecal injection on motor function and neuropathic pain, and secondary injury using both male and female rat moderate contusion SCI models.

6. Transparency, Rigor, and Reproducibility Statement

The study was designed, and data were analyzed following rigorous and reproducible scientific practices (Data S1).

Sample Size and Randomization: A total of 56 male Sprague Dawley rats (200–250 g, 7–8 weeks old) were used to generate a moderate contusion injury model for 3 studies, (1) Biodistribution (a total of 14 rats), (2) cAMP restoration by Rm‐PgP (a total of 16 rats), and (3) Efficacy of Rm‐PgP (a total of 20 rats) on secondary injury. A total of 6 rats died (four rats died during surgery and two rats died 1 day post‐injury). Sample sizes were determined based on prior power analyses, ensuring > 80% power to detect significant effects with p < 0.05. Rats were randomly assigned to experimental groups. Animals that did not survive post‐surgery were excluded from the study.

Blinding: Investigators responsible for administering treatments and conducting outcome assessments were blinded to the group allocation. Data analyses were conducted using coded identifiers.

Experimental Design and Reproducibility: The experimental procedures, including surgical induction of spinal cord injury (SCI), nanoparticle preparation, intrathecal administration, and histological analyses were performed under standardized conditions with well‐established protocols. The preparation and loading efficiency of rolipram‐loaded PgP nanoparticles (Rm‐PgP) followed a previously validated methodology. Reproducibility was assessed by confirming consistency in biodistribution, drug retention, and biological outcomes across experimental replicates.

Statistical Analysis: All statistical analyses were pre‐defined and performed using GraphPad Prism 10. Data are reported as mean ± standard deviation (STD). Group comparisons were conducted using one‐way ANOVA followed by Dunnett's multiple comparisons test. Spared tissue area across the rostrocaudal extent of the lesion was analyzed via 2‐way repeated measures ANOVA (distance from the epicenter*treatment group). Statistical significance was set at p < 0.05.

Data Integrity and Availability: All raw data, including cAMP measurements, histological images, and behavioral assessments, was verified for accuracy.

Reagent and Protocol Validation: The Rm‐PgP nanoparticle formulation was characterized using high‐performance liquid chromatography (HPLC). Antibody specificity for immunohistochemical analyses was confirmed through positive and negative controls. The stability of Rm‐PgP batches was assessed and no significant variability was observed between preparations.

Reproducibility and Ongoing Studies: The methodology and findings of this study align with previous preclinical studies investigating rolipram as a neuroprotective agent. Planned replication studies evaluating long‐term functional recovery outcomes are currently ongoing.

Author Contributions

Zhen Liao: data curation, formal analysis, investigation, visualization, writing – original draft. Jun Gao: investigation, methodology. Min Kyung Khang: investigation. Ken Webb: funding acquisition, writing – review and editing. Megan Ryan Detloff: funding acquisition, methodology, writing – review and editing. Jeoung Soo Lee: conceptualization, funding acquisition, project administration, methodology, supervision, writing – review and editing (lead).

Conflicts of Interest

Drs. Lee and Webb are co‐founders and hold equity in NeuroHope Therapeutics Inc., a small business seeking to develop PgP‐based nanotherapeutics for neurotrauma. The other authors declare no conflicts of interest.

Supporting information

Data S1: jnr70091‐sup‐0001‐supinfo.docx.

JNR-103-e70091-s001.docx (1.7MB, docx)

Acknowledgments

This work was supported by NIH/NINDS grant 5R01 NS111037 (J.S.L.), NS097880 (M.R.D.), South Carolina Spinal Cord Injury Research Fund (SC‐SCIRF) grant SCIRF #2017 B‐01, as well as the Core facility voucher program offered by the South Carolina Bioengineering Center for Regeneration and Formation of Tissues (SC BioCRAFT) at Clemson University.

Liao, Z. , Gao J., Khang M. K., Webb K., Detloff M. R., and Lee J. S.. 2025. “Rolipram‐Loaded PgP Nanotherapeutics via Intrathecal Administration Reduces Secondary Injury in a Rat Acute Moderate Contusion SCI Model.” Journal of Neuroscience Research 103, no. 11: e70091. 10.1002/jnr.70091.

Funding: This work was supported by NIH/NINDS grant 5R01 NS111037 (J.S.L.), NS097880 (M.R.D.), South Carolina Spinal Cord Injury Research Fund (SC‐SCIRF) grant SCIRF #2017 B‐01, 2023I‐01, as well as the Core facility voucher program offered by the South Carolina Bioengineering Center for Regeneration and Formation of Tissues (SC BioCRAFT) at Clemson University supported by NIH/NIGMS Grant #P30GM13195.

Edited by Lawrence S. Sherman and Christopher Anderson. Reviewed by Michel Lemay, Linqiang Tian and Tim Vanmierlo.

Data Availability Statement

The datasets generated during the current study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1: jnr70091‐sup‐0001‐supinfo.docx.

JNR-103-e70091-s001.docx (1.7MB, docx)

Data Availability Statement

The datasets generated during the current study are available from the corresponding author upon reasonable request.

Articles from Journal of Neuroscience Research are provided here courtesy of Wiley

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