Organic Anion Transporting Polypeptide (Oatp)-Mediated Transport: A Mechanism for Atorvastatin Neuroprotection in Stroke

Abstract

Bakcground:

Drug discovery for stroke is challenging as indicated by poor clinical translatability. In contrast, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (i.e., statins) improve post-stroke neurological outcomes. This property requires transport across the blood-brain barrier (BBB) via an endogenous uptake transporter (i.e., organic anion transporting polypeptide 1a4 (Oatp1a4)). Our goal was to study Oatp1a4 as a drug delivery mechanism because the BBB cannot be assumed to be completely open for all drugs in ischemic stroke.

Methods:

Male SD rats (200–250 g) were subjected to middle cerebral artery occlusion (MCAO; 90 min) followed by reperfusion for up to 7 days. Atorvastatin (20 mg/kg, i.v.) was administered 2 h following intraluminal suture removal. Involvement of Oatp-mediated transport was determined using fexofenadine (3.2 mg/kg, i.v.), a competitive Oatp inhibitor. Oatp1a4 transport activity was measured by in situ brain perfusion. Infarction volumes/brain edema ratios and neuronal nuclei (NeuN) expression were determined using TTC-stained brain tissue slices and confocal microscopy, respectively. Post-stroke functional outcomes were assessed via neurological deficit scores and rotarod analysis.

Results:

At 2 h post-MCAO, [³H]atorvastatin uptake was increased in ischemic brain tissue. A single dose of atorvastatin significantly reduced post-MCAO infarction volume, decreased brain edema ratio, increased caudoputamen NeuN expression and improved functional neurological outcomes. All MCAO positive effects of atorvastatin were attenuated by fexofenadine co-administration (i.e., an Oatp transport inhibitor).

Conclusions:

Our data demonstrate that neuroprotective effects of atorvastatin may require CNS delivery by Oatp-mediated transport at the BBB, a mechanism that persists despite increased cerebrovascular “leak” in ischemic stroke. These novel and translational findings support the utility of BBB transporters in drug delivery for neuroprotective agents.

Graphical Abstract

Brief Summary:

Brain uptake and therapeutic efficacy of atorvastatin requires a blood-brain barrier transport mechanism. These findings support transporter utility in CNS delivery of neuroprotective drugs in stroke.

Introduction

Recombinant tissue plasminogen activator (r-tPA; Alteplase) and endovascular thrombectomy (EVT) are the only FDA-approved treatments for acute ischemic stroke. These approaches involve recanalization (i.e., reperfusion); however, neither method protects against ischemia/reperfusion (I/R) injury. Complications associated with r-tPA or EVT emphasizes an unmet need for therapeutic strategies that can protect against I/R injury. In the past 30 years, many compounds have demonstrated neuroprotective properties in preclinical models, but none have advanced beyond a Phase III trial¹. Lack of translation from the bench to the clinic results from various factors including: i) lack of rigorous behavioral assessment of neurocognitive outcomes; ii) disconnects between rodent and human drug doses; and iii) failure to consider co-morbid conditions associated with stroke¹. An additional consideration is that studies examining neuroprotective drugs in stroke have not evaluated mechanisms that function simultaneously with paracellular “leak” to enable drugs to permeate the blood-brain barrier (BBB) (i.e., transporters) ². In short, we cannot assume that the BBB in stroke is fully open for all drugs everywhere and influx transporters have been shown to be effective for CNS drug delivery.

Our laboratory has shown that organic anion transporting polypeptides (OATPs in humans; Oatps in rodents) can be targeted at the BBB to facilitate therapeutic delivery into the CNS^3–5. We have focused our work on Oatp1a4, the primary drug transporting Oatp expressed at the rat BBB⁶. A human orthologue of Oatp1a4 has been identified in brain microvessels from cerebral cortical tissue and is designated OATP1A2⁶. Mechanistically, OATP1A2 and Oatp1a4 both function as facilitative diffusion transporters where the driving force across the plasma membrane is dictated by the transmembrane concentration gradient. The relevance of OATPs/Oatps to treatment of ischemic stroke is related to their ability to transport 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (i.e., statins). Amongst these drugs, atorvastatin (i.e., Lipitor®) is noteworthy due to its positive effects on neurocognitive performance following atherothrombotic stroke as measured by improved modified Rankin Scale (mRS) and Barthel index scores^7,8. In preclinical animal models of experimental ischemic stroke, atorvastatin and various other statins (i.e., simvastatin, lovastatin, rosuvastatin) have been shown to be neuroprotective as indicated by a reduction in cerebral infarction volume and/or improved neurological function^9–12. Our laboratory has shown that Oatp1a4 (in rodents) ^3–5 and OATP1A2 (in human endothelial cells) ¹³ constitute a primary mechanism for transcellular delivery of atorvastatin to the brain. Therefore, our objective is to study Oatp-mediated transport of atorvastatin in stroke and evaluate the relationship between BBB transport mechanisms and neuroprotective efficacy. This work will be conducted using the middle cerebral artery occlusion (MCAO) model of ischemic stroke and we will demonstrate that Oatp-mediated transport is an essential mechanism for atorvastatin to achieve effective CNS delivery and neuroprotective efficacy, even in the setting of increase post-stroke paracellular “leak.”

Methods

Detailed methods are presented in the Supplemental Material. All experiments were designed in accordance with the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines¹⁴. Data that support the findings of this study are available from the corresponding author upon reasonable request.

Animals and Drug Treatments

Male Sprague-Dawley (SD) rats (200–250 g; 3 months old, Envigo, Denver, CO) were used. For atorvastatin treatment experiments, animals were injected with either atorvastatin (20 mg/kg (0.4 mL/kg) in 100% ethanol, i.v.; Millipore-Sigma, St. Louis, MO) or vehicle (100% ethanol, i.v.) via the tail vein. Our experiments were conducted in healthy young males without comorbid conditions to limit experimental variability and to remove the confounding variable of estrogen effects at the BBB¹⁵. Specificity of Oatp-mediated transport was determined using fexofenadine (FEX) (3.2 mg/kg in 100% ethanol, i.v.; Millipore-Sigma), a known Oatp transport inhibitor, injected at the same time as atorvastatin.

Middle Cerebral Artery Occlusion (MCAO) Model

MCAO surgery was performed according to our previously published method¹⁶. Sham control animals underwent the same surgical procedure as MCAO animals except for insertion of the intraluminal filament. The duration of MCAO was 90 minutes for all experiments.

In situ brain perfusion

In situ brain perfusion was performed as described previously by our laboratory^4,16. These experiments were conducted following 90 minutes MCAO/2 hours reperfusion or Sham surgery, Using a slow-drive syringe pump (Harvard Apparatus Inc., Holliston, MA), [³H]atorvastatin (0.3 μCi/ml; Moravek Biochemicals Inc, Brea, Ca) or [¹⁴C]sucrose (0.3 μCi/ml; PerkinElmer Life Sciences, Boston, MA) was added to the inflowing perfusion solution. For inhibition studies, animals were perfused with Ringer’s solution containing transport inhibitor (i.e., 100 μM FEX) for 10 minutes prior to perfusion with [³H]atorvastatin.

Brain Microvessel Isolation

Microvessels were isolated from rat brain tissue using our published protocol¹⁷.

Western Blotting

Western blotting was performed as previously described⁴ with a few modifications. Polyvinylidene difluoride (PVDF) membranes were incubated overnight at 4°C with primary antibodies against Oatp1a4 (anti-Oatp1; 0.5 mg/ml at 1:500 dilution; Cat #PA5–42445; Invitrogen Life Technologies, Waltham, mA) or tubulin (anti-α tubulin; 1 mg/mL at 1:20,000 dilution; Cat #ab7291; Abcam, Cambridge, MA).

TTC staining

TTC (2,3,5-triphenyltetrazolium chloride) staining was performed in accordance with our previously published method¹⁶.

Confocal microscopy

Confocal microscopy of rat brain tissue was performed as previously described¹⁸ with a few modifications. NeuN was detected using a commercially available primary antibody (anti-NeuN, ab104224 1:500 dilution; Abcam, Boston, MA).

Behavioral Testing

Functional neurological score assessment and rotarod motor performance testing were performed in accordance with our previously published methods¹⁶. These analyses were conducted at 24 hours, 3 days and 7 days post-MCAO based on the atorvastatin elimination half-life of 14 hours. As such, effects of a single atorvastatin are unlikely beyond 7 days (12 half-lives).

Statistical Analysis

Statistical significance was determined using one-way ANOVA followed by post hoc Dunnett’s Multiple Comparison test. A value of p < 0.05 was accepted as statistically significant.

Results

Oatp1a4 Protein Expression is Reduced in Ipsilateral Cortical Tissue Following MCAO

Our experiments were designed to administer atorvastatin (20 mg/kg, i.v.) after 90 minutes MCAO followed by 2 hours of reperfusion, a translationally relevant time point within the therapeutic window for fibrinolytic therapy with r-tPA where neuroprotection is necessary (Fig. 1A). To assess transporter expression, we measured Oatp1a4 expression in ipsilateral and contralateral brain microvessels isolated from rats subjected to MCAO or in Sham-operated rats (Fig. 1B). Densitometric analysis showed decreased Oatp1a4 protein expression (normalized to α-tubulin) in ipsilateral microvessels (Fig. 1C). In contrast, Oatp1a4 protein expression did not change in contralateral cortical microvessels (Fig. 1D–E).

Figure 1: Protein Expression of Oatp1a4 in Ipsilateral and Contralateral Cerebral Cortical Microvessels after MCAO.

A: Treatment paradigm for atorvastatin (ATV) administration in male SD rats subjected to transient MCAO (90 min) followed by reperfusion up to 7 days. B: Oatp1a4 protein expression was measured by western blot analysis of brain microvessels isolated from ipsilateral cerebral cortex following transient MCAO (90 min) with reperfusion (2 h). C: Relative levels of Oatp1a4 protein expression in ipsilateral microvessels were determined by densitometric analysis and normalized to α-tubulin. D: Oatp1a4 protein expression was measured by western blot analysis of brain microvessels isolated from contralateral cerebral cortex following transient MCAO (90 min) with reperfusion (2 h). E: Relative levels of Oatp1a4 protein expression in contralateral microvessels were determined by densitometric analysis and normalized to α-tubulin. Note that each lane triplet on the depicted western blot corresponds to a microvessel sample obtained from a single experimental animal. Western blot images depict a representative blot from three individual experiments (n = 3). Quantitative results are expressed as mean ± SD from each of these three independent experiments where each western blot analyzed three individual animals (n = 3). Asterisks represent data points that were significantly different from control (** p < 0.01; ns = not significant).

Uptake of Atorvastatin to Ischemic Brain Tissue Requires Oatp-Mediated Transport

Brain accumulation of [³H]atorvastatin was studied in the presence and absence of FEX, a competitive Oatp transport inhibitor. CNS uptake of [³H]atorvastatin following 90 minutes MCAO/2 hours reperfusion was significantly increased (p < 0.01) as compared to Sham-operated controls (Fig. 2A). In the presence of FEX (100 μM), brain uptake of [³H]atorvastatin was reduced in both Sham-operated controls and in MCAO animals. Since these data only reflected total uptake of [³H]atorvastatin into whole brain tissue, we performed additional analyses to determine drug uptake into ipsilateral and contralateral cerebral cortices, respectively. As shown in Fig. 2B, blood-to-brain [³H]atorvastatin uptake was increased (p < 0.0001) into ipsilateral cortical tissue, but not into contralateral cerebral cortices, after MCAO as compared to Sham-operated controls. Within the cohort of animals subjected to MCAO, [³H]atorvastatin accumulation was also shown to be increased (p < 0.01) in ipsilateral cortex as compared to contralateral cortex. There was no difference in ipsilateral versus contralateral cortical [³H]atorvastatin uptake in Sham-operated control rats. Under both experimental conditions, blood-to-brain transport of [³H]atorvastatin was significantly reduced (p < 0.0001) in the presence of 100 μM FEX (Fig. 2B).

Figure 2: Oatp-Mediated Transport of Atorvastatin and Paracellular Diffusion of Sucrose Following MCAO.

Uptake of [³H]atorvastatin (0.3 μCi/ml) in whole brain tissue (A) and ipsilateral/contralateral brain tissue (B) from male SD rats subjected to transient MCAO (90 min) with reperfusion (2 h) and Sham-operated controls. Inhibition experiments were conducted in the presence and absence of fexofenadine (FEX; 100 μM), a competitive Oatp transport inhibitor, that was perfusion prior to administration of [³H]atorvastatin. Uptake of [³H]sucrose (0.3 μCi/ml) in whole brain tissue (C) and ipsilateral/contralateral brain tissue (D) from male SD rats subjected to transient MCAO (90 min) with reperfusion (2 h) and Sham-operated controls. Results are expressed as mean ± SD of six animals per time point (n = 6). Asterisks represent data points that were significantly different from control animals (** p < 0.01; **** p < 0.0001; ns = not significant).

A critical consideration in the interpretation of our transport data is that BBB dysfunction occurs in response to MCAO. This is emphasized by data presented in Fig. 1 and Fig. 2 where brain uptake of atorvastatin was increased following 90 minutes MCAO/2 hours reperfusion despite a reduction in BBB Oatp1a4 protein expression at the same time point. To address this issue, we measured blood-to-brain “leak” of [¹⁴C]sucrose, a small molecule vascular marker that does not cross the intact BBB¹⁹. Uptake of [¹⁴C]sucrose was significantly increased (p < 0.0001) in MCAO animals as compared to Sham-operated controls (Fig. 2C). When we compared permeability between cerebral cortices, it was observed that brain [¹⁴C]sucrose accumulation was significantly increased (p < 0.0001) in ipsilateral cortices versus contralateral cortical tissue following MCAO (Fig. 2D). There was no statistically significant change in [¹⁴C]sucrose brain uptake between ipsilateral and contralateral cortices isolated from Sham-operated controls or between contralateral cortical tissue from animals subjected to MCAO and Sham-operated controls. Despite our observation of enhanced paracellular “leak” in male SD rats after 90 minutes MCAO/2 hours reperfusion, the decrease in [³H]atorvastatin uptake in the presence of FEX demonstrates that Oatp-mediated transport may be necessary for this drug to permeate the BBB in ischemic stroke.

Neuroprotective Effects of Atorvastatin following MCAO is Dependent Upon Oatp-Mediated Transport at the BBB

To evaluate whether Oatp-mediated transport of statins is a critical mechanism that enables these drugs to be effective therapeutics for ischemic stroke, we measured brain infarction volumes and cerebral edema ratios following 90 minute MCAO/22.5 hours reperfusion (i.e., 24 hours after initiation of MCAO) and at 7 days post-MCAO. Of particular importance, no statistically significant differences in systemic blood pressure were observed across any of the treatment groups evaluated in this study. Brain infarction volumes and cerebral edema ratios were both significantly reduced (p < 0.0001) following atorvastatin administration to animals subjected to MCAO/reperfusion. (Fig 3A–C). Of translational importance, positive effects of atorvastatin in MCAO/reperfusion animals were attenuated in the presence of FEX (Fig 3A–C). In contrast, infarction volumes and edema ratios at 7 days in the MCAO and MCAO/atorvastatin/FEX groups did not show a statistically significant difference (Fig 3D–F).

Figure 3: Effect of Atorvastatin on Cerebral Infarction Volume and Edema Ratio in Male SD Rats Subjected to MCAO.

Representative TTC staining of brain tissue collected from experimental animals subjected to transient MCAO (90 minutes) with 22.5 hours reperfusion (A) or after 7 days reperfusion (B). Measurement of cerebral infarction volume in TTC-stained brain tissue after MCAO/R at 24 hours (C) or 7 days reperfusion (D). Measurement of brain edema ratio in TTC-stained brain tissue post-MCAO/R at 24 hours (E) or at 7 days (F). Results are expressed as mean ± SD of eight animals per treatment group (24 hours; n = 8) or five to six animals per treatment group (7 days; n = 5–6). Asterisks represent data points that were significantly different from control animals (**** p < 0.0001; ns = not significant).

Atorvastatin Requires Oatp-Mediated Transport to Reduce Neurological Dysfunction and Motor Impairment in the Acute Phase After MCAO

To evaluate functional neurological outcomes, we measured stroke severity using neurological deficit scores in MCAO animals at 24 hours, 3 days, and 7 days post-stroke. Neurological scores for MCAO animals that received atorvastatin (20 mg/kg i.v.) were significantly improved (1d, 3d p < 0.01; 7d p < 0.05) as compared to rats that were subjected to MCAO only (Fig 4A). During the 7 day testing period, rotarod performance was significantly enhanced with atorvastatin (20mg/kg i.v.) (3d p < 0.001; 7d p < 0.05) compared to the vehicle treated MCAO animals; however, FEX (3.2 mg/kg, i.v.) attenuated this improvement in motor function (7d p < 0.05) (Fig 4B).

Figure 4: Atorvastatin Requires Oatp-Mediated Transport to Improve Neurological Scores and Motor Performance Following MCAO.

A: Stroke severity was assessed by neurologic deficit scores in animals subjected to transient MCAO (90 minutes) with reperfusion and in Sham-operated controls at 1, 3, and 7 days. B: Motor performance was assessed by rotarod testing in animals subjected to transient MCAO (90 min) with reperfusion and in Sham-operated controls at 1, 3, and 7 days. Results are expressed as mean ± SD of six animals per treatment group (n = 6). Asterisks represent data points that were significantly different from control animals (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant).

A Single Dose Atorvastatin Protects Against Neuronal Cell Loss Following MCAO

To evaluate a specific biomarker of neuroprotection, we assessed NeuN staining in the basal ganglia of the caudoputamen, a brain region supplied by the MCA and involved in motor processing (Fig 5A). Following 24 hours reperfusion, pixel density of NeuN imaged from fresh frozen brain slices was significantly improved in response to atorvastatin treatment (20mg/kg i.v.) (p < 0.05) when compared to MCAO/R vehicle controls (Fig 5B–C). Of particular significance, atorvastatin/FEX (3.2 mg/kg, i.v.) attenuated the improvement in NeuN staining (p < 0.05) (Fig 5B–C). Overall, our data set provides novel and translational evidence for the mechanistic role of Oatp1a4 in enabling atorvastatin to confer neuroprotective effects during acute and/or subacute post-stroke recovery.

Figure 5: Acute neuroprotection in the caudoputamen of MCAO animals can be visualized using confocal microscopy.

A: Representative TTC stained brain tissue collected from experimental animal subjected to transient MCAO (90 minutes) with reperfusion (22.5 hours) and treated with atorvastatin (20 mg/kg, i.v.). The infarct core in the ipsilateral hemisphere is boxed. B: Panels depict representative images from the contralateral and ipsilateral hemispheres of Sham-operated controls animals (Sham), MCAO animals that received vehicle, ATV, or ATV/FEX. Scale bar is set to 100μm. C: Results expressed as ± SD of four independent measures of raw pixel intensity from six individual animals per treatment group (n = 6). Asterisks represent data points that were significantly different from control samples (** p < 0.01; *** p < 0.001; ns = not significant).

Discussion

Our novel study demonstrates that Oatp-mediated transport is required for atorvastatin to exert full clinically relevant neuroprotective effects in an established preclinical stroke model. Furthermore, we show that neurological outcomes following a single large dose of atorvastatin (20 mg/kg, i.v.) given 2 hours after a 90-minute occlusion are sustained for 7 days post-stroke. Atorvastatin is a transport substrate for Oatp1a4, an endogenous BBB influx transporter. Indeed, identification of other therapeutics that both confer beneficial effects in stroke and are substrates for endogenous BBB transporters presents a translational opportunity to advance stroke pharmacotherapy. This is emphasized by our recent publication showing that brain penetration and full neuroprotective effects of memantine in stroke requires functional expression of organic cation transporters (i.e., Oct1 and Oct2) at the BBB¹⁶. Coupled with this publication, the current data set supports the concept that preclinical stroke studies focused on neuroprotection and/or neuronal repair should incorporate detailed evaluation of transporters, which enable drugs to permeate the BBB and achieve effective concentrations in ischemic brain tissue (Fig. 6).

Figure 6: Interplay Between Oatp-Mediated Transport and Passive Paracellular Diffusion as Transport Mechanisms for Atorvastatin in Ischemic Stroke.

Our data demonstrates that atorvastatin requires Oatp-mediated transport to achieve efficacious concentrations following acute ischemic stroke. This leads to effective neuroprotection and an improvement in functional neurological performance during the acute phase of post-stroke recovery. When Oatp-mediated transport is impaired, positive effects of atorvastatin are attenuated despite its ability to “leak” into the brain via paracellular diffusion across the injured BBB.

Although a few studies have evaluated transporters in experimental stroke^16,20,21, there remains a knowledge gap between functional expression of BBB transport proteins and CNS delivery of stroke therapeutics. Since this objective continues to be a challenge in treatment of ischemic stroke, our laboratory’s research is focused on evaluation of BBB transport mechanisms that can be exploited for this purpose. Neuroprotective effects of atorvastatin have been reported to result from interactions in the cerebral microvasculature itself as well as in brain parenchyma. It was initially thought that statins could provide neuroprotective effects in stroke via upregulation of endothelial nitric oxide synthase (eNOS) ²²; however, studies in a murine embolic stroke model demonstrated statin neuroprotection in an eNOS-independent manner that was associated with enhanced clot lysis via endogenous tPA¹⁰. Additionally, Yang and colleagues suggested that atorvastatin interactions with neural-glial antigen 2 (NG2) in pericytes promoted neuroprotection and vascular protection in rats subjected to transient MCAO²³. Central neuroprotective effects of atorvastatin in experimental stroke are associated with binding to the GluN2B subunit of NMDA receptors²⁴, prevention of aquaporin 4 upregulation in astrocytes²⁵, or protection against neuronal apoptosis via targeting cyclic adenosine monophosphate/protein kinase A/phosphorylation of cyclic AMP response element binding protein (cAMP/PKA/p-CREB) signaling²⁶. We showed that pharmacological inhibition of Oatp1a4 by FEX decreased brain uptake and attenuated beneficial effects of atorvastatin in the MCAO model, which implies that CNS uptake of atorvastatin is required for optimal neuroprotective effects in stroke. It is critical to emphasize that drug uptake across the blood-CSF barrier (BCSFB) in unlikely to be a contributor in our data set. The BCSFB differs considerably from the BBB in terms of transporter localization and expression. Indeed, Oatp1a4 has been localized to the basolateral membrane of choroid plexus epithelial cells that comprise the BCSFB²⁷, which supports a role for solute exchange between blood and CSF; however, drug diffusion from CSF into brain extracellular fluid is slow and inefficient as a drug delivery mechanism²⁸. Overall, our present study reflects that Oatp-mediated transport is a primary mechanism for blood-to-brain delivery of atorvastatin in stroke.

It is noteworthy that Oatp-mediated transport was shown to determine blood-to-brain atorvastatin uptake despite concurrent paracellular “leak” (i.e., we cannot assume that the BBB is open to all drugs everywhere). This process persisted despite decreased Oatp1a4 protein expression in ipsilateral cortical microvessels. The short time between onset of ischemia and measurement of transporter expression (i.e., 90 min MCAO following by 2 h reperfusion) indicates that proteolytic enzymes may be involved in Oatp1a4 downregulation. Indeed, increased proteolysis mediated by matrix metalloproteinases (MMPs) is known to cause cerebral injury in stroke^29–31. Certainly, the role of MMPs in Oatp1a4 protein expression changes at the BBB requires detailed interrogation by our group. The decrease in Oatp1a4 expression also suggests that there may not be enough BBB transporter protein available to transport pharmacologically relevant concentrations of atorvastatin. We used an atorvastatin dose that produced plasma concentrations in rats that exceed K_M values for Oatp-mediated statin transport, which are reported to be in the low-to-mid micromolar range^32–34. At this dose level, transporter kinetics suggests that the 40% decrease in Oatp1a4 protein expression reported in our western blot experiments is not sufficient to greatly reduce atorvastatin transport and impair neuroprotective efficacy. Nonetheless, this observation does indicate that follow-up experiments designed to determine the time window post-stroke when Oatp-mediated transport can be most effectively exploited for CNS statin delivery will be considered. An additional consideration in the translatability of our work is the potential for species differences in transport properties between Oatp1a4 and its human orthologue OATP1A2. This issue is best exemplified by of Liu and colleagues who showed OATP1A2-mediated transport of zolmitriptan but lack of transport of multiple other triptans by Oatp1a4³⁵. We agree that rodent models are not appropriate for examination of triptan transport properties; however, this is not the case for statins. We have conclusively demonstrated that atorvastatin is a transport substrate for both Oatp1a4 and OATP1A2^3,4,13. Taken together, these data prove the translational potential of our in vivo rodent studies on Oatp1a4-mediated transport in stroke.

Statins remain formulated for oral administration only, a factor that limits their use to those patients that can swallow during acute post-stroke recovery. Dysphagia is experienced by approximately 50% of stroke patients^36,37. We incorporated this issue into our research design by administering atorvastatin intravenously. This strategy improved indices and biomarkers of neuroprotection (i.e., cerebral infarction volume, brain edema ratios, NeuN staining) at 24 h post-MCAO; however, improvements in functional neurological outcomes persisted at post-stroke time points up to 7 days. This apparent disconnect can be explained by MCA anatomy within the SD rat strain. As noted by Biose and colleagues, differences in cerebral infarction volume at 7 days post-MCAO may arise, in part, due to variability in MCA branching that is known to occur in SD rats³⁸, which can cause increased blood flow and a concurrent reduction in infarction volume despite monofilament occlusion³⁹. Indeed, variability in MCA branching also exists within cohorts of human subjects⁴⁰, which emphasizes the translational applicability of the SD rat strain to preclinical stroke research. Coupled with the elimination half-life of atorvastatin (i.e., 14 hours), which indicates that a single dose will be completely cleared by 7 days, and the persistence of a subcortical infarction at 24 h and 7 days post-MCAO, anatomical MCA variability can explain the discordance between measurement of indices of neuroprotection and our behavioral studies. Recent studies suggest that differences in vascular structure including collaterals may influence stroke evolution in animal models and human patients^41,42. Furthermore, factors including age, hypertension, and metabolic disease modulate the complex interactions between systemic biology and the ischemia-damaged neurovascular unit⁴³. Our present study in young SD rats demonstrates the fundamental importance of BBB transporters in drug delivery after cerebral ischemia. Further studies are warranted to rigorously assess these mechanisms in other animal models that mimic collateral diversity, aging, and comorbidities in clinical stroke.

It is generally accepted that administration of a statin early following an ischemic stroke improves functional neurological outcomes^44,45. In contrast, Yoshimura and colleagues reported that early statin therapy within 24 hours of hospital admission did not show any superior improvement in neurocognitive outcomes as compared to patients where pharmacotherapy with statins was received at 7 days post-stroke⁴⁶. When interpreting these results, it is critical to point out that the study population included ischemic stroke patients with small infarctions, which could have caused the inability of the investigators to detect an improvement in the mRS at 3 months (i.e., the primary endpoint of the study) ^46,47. Nonetheless, the totality of clinical stroke literature suggests that early administration of statins to stroke patients is safe and can promote improved neurological and motor outcomes during recovery.

Overall, this study has identified Oatp-mediated transport at the BBB as a critical process that facilitates atorvastatin delivery to the ischemic brain, thereby providing this drug with an opportunity to provide beneficial therapeutic effects during acute post-stroke recovery. This work advances current knowledge on the BBB in stroke by demonstrating that both selective transport (i.e., endogenous transporter proteins) and paracellular diffusion (i.e., “leak”) are critical determinants of drug delivery to the ischemic brain. Furthermore, results presented in this paper strengthen evidence generated by our laboratory that endogenous BBB transporters can be targeted for delivery of therapeutic drugs, thereby providing a platform for development of novel pharmacological treatment strategies for ischemic stroke.

Nonstandard Abbreviations

ARRIVE

Animal Research: Reporting In Vivo Experiments

BBB

blood-brain barrier

BCSFB

blood-cerebrospinal fluid barrier

eNOS

endothelial nitric oxide synthase

EVT

endovascular thrombectomy

FEX

fexofenadine

HMG-CoA

3-hydroxyl-3-methylglutaryl coenzyme A

I/R

ischemia/reperfusion

MCA

middle cerebral artery

MCAO

middle cerebral artery occlusion

MMP

matrix metalloproteinase

mRS

modified Rankin scale

NG2

neural-glial antigen 2

OATP

organic anion transporting polypeptide

Oct

organic cation transporter

PVDF

polyvinylidene difluoride

SD

Sprague-Dawley

TTC

2,3,5-triphenyltetrazolium chloride