Effects of postconditioning on neurogenesis and angiogenesis during the recovery phase after focal cerebral ischemia

Abstract

Background and Purpose

Postconditioning may be a clinically feasible way to protect the brain after a stroke. However, its effects during the recovery phase post-stroke remain to be fully elucidated. Here, we examine the hypothesis that ischemic postconditioning amplifies neurogenesis and angiogenesis during stroke recovery.

Methods

Male Sprague-Dawley rats were subjected to 100min transient middle cerebral artery occlusion (MCAO) or postconditioning (100min MCAO plus 10min reperfusion plus 10 min reocclusion). After 2 weeks, infarct volumes, behavioral outcomes and immunohistochemical markers of neurogenesis and angiogenesis were quantified.

Results

Postconditioning significantly reduced infarction and improved neurologic outcomes. Concomitantly, brains subjected to postconditioning showed an increase in doublecortin/BrdU and collagen-IV/Ki67 positive cells.

Conclusions

These results suggest that therapeutic effects of postconditioning may involve the promotion of neurogenesis and angiogenic remodeling during the recovery phase after focal cerebral ischemia.

INTRODUCTION

In a recent consensus workshop, leaders in the innate tolerance field identified postconditioning as a potentially powerful and clinically relevant approach for stroke¹. Experimental studies suggest that ischemic postconditioning interferes with cell death mechanisms and reduces infarction during the acute phase after focal cerebral ischemia².

In previous studies two key animal models of ischemic postconditioning were mainly used. The permanent distal occlusion of the middle cerebral artery followed by a series of occlusion of both common carotid arteries (CCAs) and a 100 minutes middle cerebral artery occlusion followed by 10 minutes of reperfusion and 10 minutes of reocclusion. Ischemic postconditioning was also investigated in animals subjected to global ischemia induced by occlusion of the CCAs and of the two vertebral arteries, 4-vessel occlusion, followed by different cycles of non-injurious CCA occlusion². Taken together, these studies suggest that postconditioning may offer positive protective effects during the acute phase of stroke. However, how postconditioning may provide longer lasting benefits during stroke recovery remains unclear³.

During the recovery phase after stroke, an increase in angiogenesis and neurogenesis occurs. It has been proposed that these neurovascular responses provide the required signals and substrates for plasticity and remodeling as damaged brain tissue attempt to reorganize and recover⁴. In this proof-of-concept study, we used a rat model of focal cerebral ischemia to assess the hypothesis that the beneficial effects of postconditioning may involve the amplification of neurogenesis and angiogenesis during the delayed periods after initial injury.

METHODS

Middle cerebral artery occlusion and ischemic postconditioning

All experiments were performed following protocols approved by Massachusetts General Hospital Institutional Animal Care and Use Committee in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. We used two groups of stroke animals, one group exposed to postconditioning, and the other group exposed to standard post-stroke care. Transient focal ischemia was induced by introducing a 5.0 surgical monofilament nylon suture (Doccol) into the middle cerebral artery (MCA) for 100 minutes in male Sprague–Dawley rats. Ischemic postconditioning was induced as previously described⁵. Briefly the postconditioning technique involves 100 minutes of occlusion, then reperfusion was established for 10 minutes after which the MCA was reoccluded for another 10 minutes⁵.

To analyze cell renewal processes, BrdU was dissolved in PBS at 10mg/ml and injected IP to a dose of 50mg/kg every second day for two weeks. Animals were recovered for 14 days. Neurological scores were graded on a scale of 0 to 4⁶, with a higher score indicating more severe sensory-motor deficits. All procedures and measurements were performed in a blinded and randomized fashion. Physiological parameters and rCBF did not change between the two groups (data not shown).

Histology and immunohistochemistry

Infarction volumes were quantified on Nissl-stained sections using the “indirect” morphometric method. Immunohistochemistry was performed as described before⁶. To assess microvessel remodeling, double-labeling of anti-type IV Collagen (1:10, SouthernBiotech) with anti-Ki67 (1:500, Abcam) (a general cell proliferation marker) was pursued as a surrogate marker of angiogenic-related events. To study neurogenic-related events, we double-stained anti-DCX (1:100, Abcam) with anti BrdU (1:50, Invitrogen) as a surrogate marker of neurogenesis.

To clarify that Ki67 positive cells are not proliferating microglia/macrophages we double-stained Ki67and Iba1.

Statistical analysis

Values are expressed as mean ± SD. Infarct volumes and cell counts of immunopositive cells were assessed with Student's t-test. Neurological outcomes were analyzed using Mann Whitney test. P values of p<0.05 were considered statistically significant.

RESULTS

At 2 wks, Nissl staining revealed well-defined infarcts in all control animals subjected to 100 minutes of MCAO (181.9±17.68 mm3, n=8). In rats that were subjected to ischemic postconditioning, infarct volumes were markedly reduced (132.8±10.74 mm3, n=9, P<0.05, Fig 1A). Postconditioning had also a positive effect on neurological outcomes. Rats treated with postconditioning had significantly better scores (0.4±0.4, n=9) compared to controls (1.2±0.7, n=8, Fig.1B).

Figure 1. Ischemic volume and behavioral outcomes.

(A) Ischemic volume was significantly smaller in postconditioning group. (B) Neuroscores were decreased after postconditioning treatment. *P<0.05. Mean ± S.D.

Markers of neurogenesis were analyzed in the peri-infarct regions at 2 wks. Immunohistochemistry demonstrated that enhanced signals for DCX/BrdU were detected in the ipsilateral hemisphere. Compared to untreated controls (19.67±5.5 positive cells/mm2), DCX/BrdU-positive cells appeared to be increased in animals subjected to ischemic postconditioning (53±10.5 positive cells/mm2) (Fig 2).

Figure 2. Ischemic postconditioning increased neurogenesis.

DCX/Brdu cell positive number increased in the peri-infarct area after postconditioning treatment indicating that postconditioning activates neurogenesis. *P<0.05. Mean ± S.D.

As a marker for angiogenesis, immunostaining was performed to quantify microvessels that were double-positive for collagen-IV and Ki67. As expected, peri-infact regions appeared to show an increase in microvessels. The density of collagen-IV-Ki67 microvessels was significantly higher in the postconditioning group (80.74±22.49 positive cells/mm2) compared with controls (30.09±14.07 positive cells/mm2). We did not detect any increase in Ki67/Iba1 double staining in postconditioning group compared to controls, suggesting that microglia may not provide a large contribution to our signals (Fig 3).

Figure 3. Ischemic postconditioning increased angiogenesis.

(A) The cell numbers co-expressing collagen IV and anti-Ki67/mm² in the peri-infarct area were increased in postconditioning group. (B) No difference was observed in co-expression of Ki67 and Iba1. *P<0.05. Mean ± S.D.

DISCUSSION

After brain injury, tolerance mechanisms are activated as part of the endogenous neuroprotective program^{7, 8}. It is increasingly recognized that finding ways to boost these endogenous mechanisms may provide novel avenues for stroke therapy^1,9. Accumulating studies in various experimental models now suggest that ischemic postconditioning may provide acute protection¹⁰. But what may be missing is a full understanding of the mechanisms responsible for postconditioining and long-term neuroprotection. In this proof-of-concept study, we used a rat model of transient focal ischemia to confirm that beneficial effects of postconditioning may last for up to 2 wks. Our main goal in this study was to show that potential benefits of postconditioning were accompanied by augmentation of neurogenic and angiogenic-related markers in recovering brain tissue.

Ischemic brain insults potently stimulate progenitor proliferation in both the SGZ and SVZ of adult rodents¹¹. Neuron progenitors are then able to migrate to injury sites, perhaps as part an endogenous repair response after stroke and brain injury. Similarly, angiogenesis, i.e. the growth of blood vessels from the existing vasculature, may also contribute to the recovery phase after stroke. Pro-angiogenic genes are upregulated within minutes of the onset of cerebral ischemia in rodents¹², and within the peri-infact zone, angiogenesis may significantly participate in neurovascular remodeling and recovery. Increasingly, it has been suggested that delayed effects of many stroke therapies may involve the augmentation of neurogenesis and angiogenesis¹³. Our findings here raise the possibility that post-conditioning may also recruit these endogenous protective mechanisms.

Taken together, the present study suggests that beneficial effects of ischemic postconditioning can be maintained up to 2 wks post-ischemia, and the underlying mechanisms may be consistent with improvements in post-stroke neurogenesis and angiogenesis. However, there are several caveats to keep in mind. First, we only examined a single protocol for postconditioning. Whether outcomes can be further improved with different “doses” and “timing” remains to be tested. Extending the present “single” and “100-minute post-stroke” postconditioning regimen closer to or even beyond the 4.5 hours of tPA window may be an important next step. Second, besides neuronal and vascular cells, other cell types may also be involved. For example, immune cells such as macrophages and microglia are known to contribute to neurogenic and angiogenic phenomenon. In our study, Ki67 positive cells did not double-stain for Iba1, suggesting that postconditioning may not affect this response in our model. However, further studies to assess effects of postconditioning on immune profiles are warranted. Third, sustained clinical benefit must be more rigorously explored. Two weeks endpoints may be a reasonable timeframe for exploring delayed effects in experimental models. But for translational assurance, longer-term studies are needed and more behavioral studies specific for long term outcomes should be considered. Finally, this remains a proof-of-concept study and our results cannot prove causality. Whether the markers of neurogenesis and angiogenesis are causative or correlational must be carefully assessed in future gain and loss-of-function experiments. Postconditioning may provide a promising therapeutic approach for stroke. Continued investigation into its potential mechanisms is warranted.