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the Department of Neurosurgery (C.M.M., F.Y., T.N., P.H.C.), Department of Neurology and Neurological Sciences and Program in Neurosciences, Stanford University School of Medicine, Stanford, Calif
Maxygen, Inc (S.J.L.), Redwood City, Calif.
Abstract
Background and Purpose— Compelling evidence supporting the role of inflammation in the development of cerebral infarction has focused attention on the potential of antiinflammatory treatment strategies for stroke. Interferon (IFN)-, an immunomodulatory agent approved for treatment of multiple sclerosis, is being evaluated in a phase I clinical trial in acute ischemic stroke. In the present study, we evaluated the effects of wild-type rat IFN- and its pegylated counterpart (PEG-IFN-) in a model of focal ischemia and reperfusion.
Methods— After 60 minutes of middle cerebral artery occlusion, rats (n=12/group) were treated with IV tail injections of 8 or 16 μg of IFN- in 300 μL of PBS once daily for 3 or 7 days or with IV or SC injections of PEG-IFN- for 1 day. The animals were assessed daily for weight and for neurological findings. Additional animals underwent complete hematology and chemistry profiles, as well as complete multiorgan necropsy studies. All of the brain tissue was evaluated for assessment of infarct areas, neutrophil infiltration, and presence of hemorrhagic transformations.
Results— IFN- and PEG-IFN- failed to protect against experimental ischemic brain injury as assessed by histopathology and neurological outcome. Furthermore, IFN- treatment was associated with significant weight loss and alterations in hematology and chemistry profiles.
Conclusions— Our results suggest that additional preclinical studies are warranted.
Key Words: inflammation interferon ischemia neuroprotection
Introduction
Inflammation plays a key role in the pathophysiology of ischemic stroke, and several antiinflammatory treatment strategies have been shown to mitigate secondary brain injury after experimental cerebral ischemia1. Interferon (IFN)-, a cytokine with immunomodulatory properties, was approved by the US Food and Drug Administration for treatment of relapsing-remitting multiple sclerosis >10 years ago.2 A National Institute of Neurological Disorders and Stroke-sponsored phase I clinical trial is underway to determine the safety, tolerability profile, and optimal administration route of IFN- in patients with acute ischemic stroke. The rationale for use of IFN- in stroke patients comes from a limited number of animal studies in which systemic administration of IFN- before or after (4 to 6 hours after) stroke onset reduced infarct volumes at 24 hours3 and after 3 weeks of reperfusion.4 The neuroprotective effects of IFN- are associated with a reduction in neutrophil infiltration and decreased blood–brain barrier (BBB) disruption,4–6 as well as with the promotion of cell survival factors.7 However, the exact mechanisms through which IFN- might moderate the development of ischemic stroke remain unclear. One question is whether the drug can directly access the central nervous system, because it does not penetrate the BBB well unless the barrier is substantially compromised during the ischemic insult. To date, only 2 animal stroke models have been reported that evaluate the efficacy of IFN-: (1) transient (1 hour) occlusion of the middle cerebral artery (MCA) via direct placement of a microvascular clip,4 and (2) permanent MCA occlusion (MCAO) by injection of an autologous clot.3 Both of these models result in substantial BBB breakdown and cerebral edema.
In the present study, we examined the effects of wild-type rat IFN- and its pegylated counterpart (PEG-IFN-) in a focal cerebral ischemia model (intraluminal suture method8). This model has the advantage of being less invasive, because it does not require a craniotomy, yet allows drug effects to be evaluated after reperfusion and has been successfully used to test a variety of neuroprotective agents. In our studies, neither IFN- nor PEG-IFN- was neuroprotective, as determined by histopathology and behavioral outcomes, at doses reported previously to reduce ischemic lesion volume. Furthermore, some IFN-–treated animals developed considerable weight loss and alterations in hematology and chemistry profiles, although the clinical significance of these changes is not clear.
Methods
Focal Cerebral Ischemia
All of the procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Administrative Panel on Laboratory Animal Care of Stanford University. Adult male Sprague-Dawley rats (275 to 300 g) were subjected to transient focal ischemia by an intraluminal MCA blockade.8 Rectal temperature was maintained at 37±0.5°C. After 60 minutes of MCAO, blood flow was restored by suture withdrawal.
Drug Protocol
Rat IFN- was purchased from U-CyTech (Utrecht, the Netherlands). IFN- pegylation was done as described previously9 (with modifications). The pegylated compound was diluted, filtered, and purified by cation exchange and size exclusion chromatography. Protein-containing fractions were analyzed on a 4% to 12% Bis- Tris-SDS-PAGE gel using the 2-(N-morpholino)ethanesulfonic acid buffer system (Novex). Monopegylated IFN- fractions were pooled, the concentration measured, samples filtered, and stored at –70°C in PBS with 3 mg/mL rat serum albumin (Sigma). Animals were treated with IV tail injections of 8 or 16 μg of rat IFN- in 300 μL of PBS once daily for 3 or 7 days or with IV or SC injections of PEG-IFN- for 1 day. Those receiving drug for only 1 or 3 days were injected with formulation buffer for the remaining days. Animals were randomized into 9 groups (n=12/group): Sham control (intraluminal suture not advanced), permanent MCAO, formulation buffer, IFN- (8 μg for 3 days, 8 μg for 7 days, 16 μg for 3 days, and 16 μg for 7 days), PEG-IFN- (16 μg for 1 day IV and 16 μg for 1 day SC). Additional animals (n=3/group) treated with IV buffer or IFN- (16 μg for 3 days) were used for hematology and chemistry profiles and for complete multiorgan necropsy studies. Investigators were blinded to the treatment groups.
Immunohistochemistry
The rats were anesthetized with an isoflurane overdose on day 8. Each brain was sliced coronally at 2-mm intervals, soaked (10 minutes) in 2% 2,3,5-triphenyltetrazolium chloride (TTC) in 0.1 mol/L PBS (pH 7.4), and fixed in 10% buffered formalin. After paraffin embedding, 6-μm–thick sections were stained with hematoxylin/eosin. Infarct areas were quantified by an image analysis system (Bio-Rad Laboratories). Adjacent sections were incubated with blocking solution and reacted with an antimyeloperoxidase (MPO) antibody (1:100 dilution), which was detected using a Vector-ABC kit and colorized with Vector-VIP (Vector Laboratories). Diaminobenzidine-enhanced Perl’s iron staining was carried out by incubation in 1% KFeCN/1% HCl followed by methyl green counterstain.
Postoperative Care
The animals were assessed daily for weight and for neurological findings using a previously reported neurological grading scale.10 Neurological assessment included level of consciousness, sensorimotor function, gait, grooming, eating/drinking, and exploratory behavior. A score of zero indicated no neurological deficits.
Statistical Analyses
Statistical analyses were done with 1-way ANOVA for continuous data and with nonparametric tests for noncontinuous data. All of the data were expressed as mean±SEM; a P value <0.05 was considered significant.
Results
Treatment with IFN- or PEG-IFN- failed to protect against ischemic brain injury (Figure 1A), as determined from TTC- stained sections (confirmed by hematoxylin/eosin; Figure 2), and did not improve neurological scores (Figure 1B) compared with buffer-treated animals. Furthermore, although all of the animals exhibited weight reduction in the first 2 postoperative days, IFN-–treated animals (16 μg for 3 days) weighed, on average, 45 g less than buffer-treated rats by day 7 (P0.032; Figure 1C). At 1 week, animals in the IFN- 8-μg 3-day and IFN- 16-μg 7-day groups also showed significant weight loss, weighing 32 and 33 g less, respectively, than buffer-treated animals (P<0.05), which was also observed in sham controls treated with 8 μg of IFN- for 3 days (pilot study data not shown).
Results from the necropsy studies showed similar pathology on all of the IFN-–treated animals: some mild hepatic atrophy with complete loss of glycogen stores, minimal to mild multifocal accumulations of mixed inflammatory cells, scattered apoptotic bodies, mitotic figure, and binucleate and multinucleate hepatocytes. Complete multiorgan necropsy/histopathology showed that all of other tissues examined, including kidney, were considered to be within normal limits. Complete hematology and chemistry profiles showed that IFN-–treated animals had consistently low white blood cell counts (<10 K/μL; normal range [nr], 13.2 to 16.6), high red blood cell counts (>8.6 mol/L/μL; nr, 5.8 to 8.4), high hemoglobin values (>16.6 g/dL; nr, 13.3 to 16.1), high hematocrit (>51.8%; nr, 41.3 to 49.3), and high glucose levels (>134 mg/dL; nr, 70 to 126), whereas buffer-treated animals were within normal range. Two of 3 IFN-–treated animals also showed a high blood urea nitrogen:creatinine ratio (>77 mg/dL; nr, 15.7 to 50). It is important to note that, despite being observed eating and drinking, animals exhibiting significant weight loss by day 3 received additional subcutaneous fluids daily.
Qualitative examination of brain tissue sections showed a very low incidence of MPO-positive cells in IFN-–treated animals (Figure 2). Intraparenchymal Perl’s iron staining (present in hemorrhagic transformations) was similarly limited in both buffer- and IFN-–treated animals and, when present, was restricted mostly to the entry point of the MCA, suggesting that mild mechanical damage to the occluded vessel may have occurred in some animals (data not shown).
Discussion
As a potent immunomodulatory agent, IFN- has the potential to counteract detrimental inflammatory events after ischemic brain injury. In the present study, however, both IFN- and its pegylated counterpart failed to confer neuroprotection 1 week after stroke onset. Our results contradict previous studies in which IFN- treatment after cerebral ischemia/reperfusion resulted in reduced infarct volumes up to 3 weeks after stroke onset, as determined from MRI.4 One possible explanation is that the BBB was not sufficiently disrupted in our model to allow IFN- to reach the affected tissue. However, there is no direct evidence available to suggest that IFN- exerts any pharmacological effects within the brain parenchyma. As an antiinflammatory, IFN- may work, in part, by promoting the release of soluble adhesion molecules from cerebral endothelial cells. These soluble factors can then bind to antigens in circulating leukocytes, thereby reducing their ability to traffic across the BBB.11,12 This hypothesis is supported by Veldhuis et al,4,6 who showed that IFN- treatment decreased neutrophil infiltration and attenuated BBB disruption after ischemia/reperfusion. In our study, we found no differences in Perl’s iron staining between buffer- and INF-–treated animals. Although we found a qualitative reduction in MPO immunoreactivity in the latter, our observations were limited to a single time point late in the evolution of the infarct and, thus, may not reflect the neutrophil infiltration pattern earlier in the inflammatory process.
In addition to the lack of neuroprotection, we found that IFN- treatment was associated with significant weight loss and alterations in hematology and chemistry profiles. None of the necropsy findings explain the severe weight loss; however, the hepatocellular atrophy and apoptosis could be secondary to weight loss. The inflammation observed in the liver tissue was not significant enough to cause destruction of hepatic mass. In fact, small foci of inflammatory cells are often noted in livers secondary to normal gut flora seeding the liver through the portal system. Mitotic figures in hepatocytes are not very common, and their significance in these animals is unclear.
Although the clinical implications of the weight loss and alterations in hematology and chemistry profiles are not clear, it is worth noting that 2 alerts warning of hepatic injury associated with IFN- treatment in multiple sclerosis patients have already been issued.13–15 Our results suggest that additional preclinical studies are desirable before more advanced clinical testing of IFN- in stroke treatment.
Acknowledgments
This work was supported by Maxygen, Inc, and by an American Heart Association Bugher Foundation Award (to P.H.C.). The authors thank Dona Bouley for necropsy studies, Stephanie Murphy for expert advice, Cheryl Christensen for editorial assistance, and Elizabeth Hoyte for figure preparation.
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