Intraluminal Thread Technique Research Articles

The selective oestrogen receptor modulator, bazedoxifene, mimics the neuroprotective effect of 17β-oestradiol in diabetic ischaemic stroke by modulating oestrogen receptor expression and the MAPK/ERK1/2 signalling pathway.

Because neuroprotection in stroke should be revisited in the era of recanalisation, the present study analysed the potential neuroprotective effect of the selective oestrogen receptor modulator, bazedoxifene acetate (BZA), in an animal model of diabetic ischaemic stroke that mimics thrombectomy combined with adjuvant administration of a putative neuroprotectant. Four weeks after induction of diabetes (40mgkg-1 streptozotocin, i.p.), male Wistar rats were subjected to transient middle cerebral artery occlusion (intraluminal thread technique, 60minutes) and assigned to one of three groups treated with either: vehicle, BZA (3mgkg-1 day-1 , i.p.) or 17β-oestradiol (E2 ) (100μgkg-1 day-1 , i.p.). At 24hours post-ischaemia-reperfusion, brain damage (neurofunctional score, infarct size and apoptosis), expression of oestrogen receptors (ER)α, ERβ and G protein-coupled oestrogen receptor), and activity of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)1/2 and phosphoinositide 3-kinase/Akt pathways were analysed. At 24hours after the ischaemic insult, both BZA- and E2 -treated animals showed lower brain damage in terms of improved neurofunctional condition, decreased infarct size and decreased apoptotic cell death. Ischaemia-reperfusion induced a significant decrease in ERα and ERβ expression without affecting that of G protein-coupled oestrogen receptor, whereas BZA and E2 reversed such a decrease. The ischaemic insult up-regulated the activity of both the MAPK/ERK1/2 and phosphoinositide 3-kinase/Akt pathways; BZA and E2 attenuated the increased activity of the ERK1/2 pathway, without affecting that of the Akt pathway. The results of the present study lend further support to the consideration of BZA as an effective and safer alternative overcoming the drawbacks of E2 with respect to improving diabetic ischaemic stroke outcome after successful reperfusion.

Molecular mechanisms mediating the neuroprotective role of the selective estrogen receptor modulator, bazedoxifene, in acute ischemic stroke: A comparative study with 17β-estradiol

As the knowledge on the estrogenic system in the brain grows, the possibilities to modulate it in order to afford further neuroprotection in brain damaging disorders so do it. We have previously demonstrated the ability of the selective estrogen receptor modulator, bazedoxifene (BZA), to reduce experimental ischemic brain damage. The present study has been designed to gain insight into the molecular mechanisms involved in such a neuroprotective action by investigating: 1) stroke-induced apoptotic cell death; 2) expression of estrogen receptors (ER) ERα, ERβ and the G-protein coupled estrogen receptor (GPER); and 3) modulation of MAPK/ERK1/2 and PI3K/Akt signaling pathways. For comparison, a parallel study was done with 17β-estradiol (E2)-treated animals. Male Wistar rats subject to transient right middle cerebral artery occlusion (tMCAO, intraluminal thread technique, 60min), were distributed in vehicle-, BZA- (20.7±2.1ng/mL in plasma) and E2- (45.6±7.8pg/mL in plasma) treated groups. At 24h from the onset of tMCAO, RT-PCR, Western blot and histochemical analysis were performed on brain tissue samples. Ischemia-reperfusion per se increased apoptosis as assessed by both caspase-3 activity and TUNEL-positive cell counts, which were reversed by both BZA and E2. ERα and ERβ expression, but not that of GPER, was reduced by the ischemic insult. BZA and E2 had different effects: while BZA increased both ERα and ERβ expression, E2 increased ERα expression but did not change that of ERβ. Both MAPK/ERK1/2 and PI3K/Akt pathways were stimulated under ischemic conditions. While BZA strongly reduced the increased p-ERK1/2 levels, E2 did not. Neither BZA nor E2 modified ischemia-induced increase in p-Akt levels. These results show that modulation of ERα and ERβ expression, as well as of the ERK1/2 signaling pathway accounts, at least in part, for the inhibitory effect of BZA on the stroke-induced apoptotic cell death. This lends mechanistic support to the consideration of BZA as a potential neuroprotective drug in acute ischemic stroke treatment.

TDZD-8 pre-treatment in transient middle cerebral artery occlusion

There is an unmet clinical need to develop neuroprotective agents for cerebrovascular procedures requiring transient cerebral artery occlusion. This study aims to investigate the effects of a single pre-treatment dose of 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), a glycogen synthase kinase-3β (GSK-3β) inhibitor, in a transient focal cerebral ischemia model. Twenty-eight male adult Wistar rats were subjected to right middle cerebral artery (MCA) occlusion via intraluminal thread technique for 60min under continuously cortical perfusion monitoring by laser-Doppler flowmetry. Rats were divided into two groups: control or treatment groups. In the treated group, TDZD-8 (5mg/kg; intravenously) was administered 10min before the onset of the MCA ischemia. At 24-h reperfusion, the following parameters were evaluated: neurological deficits, brain infarct volume, ipsilateral hemispheric oedema, neuron specific enolase (NSE) plasma levels, parenchyma histology (H–E staining), Fluoro-Jade positive neurons, p-Akt and total Akt expression by western blot analysis and p-Akt-positive nuclei by immunohistochemistry. Infarct volume (P<0.001) and neurological deficits severity (P<0.001) were reduced in TDZD-8 treated group. TDZD-8 attenuated hemispheric oedema (P<0.001), prevented the NSE plasma level increase (P<0.001) and diminished the number of degenerated neurons in the infarct area (P<0.001), as shown by Fluoro-Jade staining. TDZD-8 treated rats showed few signs of perivascular oedema when compared to control group. No variations in total Akt and p-Akt expression were observed; instead immunohistochemistry showed increased p-Akt nucleus translocation in TDZD-8 treated rats (P<0.05). TDZD-8 is a potential pre-treatment intraoperative drug to prevent neuronal injury induced by transitory artery occlusion during cerebrovascular procedures.

The selective estrogen receptor modulator, bazedoxifene, reduces ischemic brain damage in male rat

While the estrogen treatment of stroke is under debate, selective estrogen receptor modulators (SERMs) arise as a promising alternative. We hypothesize that bazedoxifene (acetate, BZA), a third generation SERM approved for the treatment of postmenopausal osteoporosis, reduces ischemic brain damage in a rat model of transient focal cerebral ischemia. For comparative purposes, the neuroprotective effect of 17β-estradiol (E2) has also been assessed. Male Wistar rats underwent 60min middle cerebral artery occlusion (intraluminal thread technique), and grouped according to treatment: vehicle-, E2- and BZA-treated rats. Optimal plasma concentrations of E2 (45.6±7.8pg/ml) and BZA (20.7±2.1ng/ml) were achieved 4h after onset of ischemia, and maintained until the end of the procedure (24h). Neurofunctional score and volume of the damaged brain regions were the main end points. At 24h after ischemia–reperfusion, neurofunctional examination of the animals did not show significant differences among the three experimental groups. By contrast, both E2- and BZA-treated groups showed significantly lower total infarct volumes, BZA acting mainly in the cortical region and E2 acting mainly at the subcortical level. Our results demonstrate that: (1) E2 at physiological plasma levels in female rats is neuroprotective in male rats when given at the acute stage of the ischemic challenge and (2) BZA at clinically relevant plasma levels mimics the neuroprotective action of E2 and could be, therefore, a candidate in stroke treatment.

Neuroprotective effects of erythropoietin pretreatment in a rodent model of transient middle cerebral artery occlusion

OBJECT.: There is an unmet clinical need to develop neuroprotective agents for neurosurgical and endovascular procedures that require transient cerebral artery occlusion. The aim in this study was to explore the effects of a single dose of recombinant human erythropoietin (rhEPO) before middle cerebral artery (MCA) occlusion in a focal cerebral ischemia/reperfusion model. Twenty-eight adult male Wistar rats were subjected to right MCA occlusion via the intraluminal thread technique for 60 minutes under continuous cortical perfusion monitoring by laser Doppler flowmetry. Rats were divided into 2 groups: control and treatment. In the treated group, rhEPO (1000 IU/kg intravenously) was administered 10 minutes before the onset of the MCA ischemia. At 24-hour reperfusion, animals were examined for neurological deficits, blood samples were collected, and animals were killed. The following parameters were evaluated: brain infarct volume, ipsilateral hemispheric edema, neuron-specific enolase plasma levels, parenchyma histological features (H & E staining), Fluoro-Jade-positive neurons, p-Akt and total Akt expression by Western blot analysis, and p-Akt-positive nuclei by immunohistochemical investigation. Infarct volume and Fluoro-Jade staining of degenerating neurons in the infarct area did not vary between groups. The severity of neurological deficit (p < 0.001), amount of brain edema (78% reduction in treatment group, p < 0.001), and neuron-specific enolase plasma levels (p < 0.001) were reduced in the treatment group. Perivascular edema was histologically less marked in the treatment group. No variations in the expression or localization of p-Akt were seen. Administration of rhEPO before the onset of 60-minute transient MCA ischemia protected the brain from this insult. It is unlikely that rhEPO pretreatment leads to direct neuronal antiapoptotic effects, as supported by the lack of Akt activation, and its benefits are most probably related to an indirect effect on brain edema as a consequence of blood-brain barrier preservation. Although research on EPO derivatives is increasing, rhEPO acts through distinct neuroprotective pathways and its clinical safety profile is well known. Clinically available rhEPO is a potential therapy for prevention of neuronal injury induced by transitory artery occlusion during neurovascular procedures.

Protective effect of treatment with low-dose gliclazide in a model of middle cerebral artery occlusion and reperfusion in rats

The aim of this study was to explore the expression of sulfonylurea receptor 1 (SUR1), the regulatory subunit of the NCCa-ATP channel, and to investigate the protective effects of gliclazide following middle cerebral artery occlusion (MCAO)/reperfusion in male Wistar rats. Adult rats underwent 2h of the left MCAO using the intraluminal thread technique before reperfusion. The core areas of the infarct at different reperfusion time points were examined for the mRNA level and protein expression of SUR1 using reverse transcription-polymerase chain reaction (RT-PCR) and western blotting respectively. Gliclazide was administered intravenously into the right jugular vein for 12h simultaneously with the reperfusion. The number of apoptotic cells was determined using the TUNEL assay. The neurological functional deficits were evaluated using Bederson׳s test, and the cerebral infarction volume was visualized with TTC staining. We found up-regulation of SUR1 mRNA and protein levels in ischemic infarct tissues after reperfusion following MCAO, and SUR1 mRNA and protein were maximally upregulated 8–12h after a 2-hour ischemia. The treatment with low-dose of gliclazide reduced the total number of TUNEL-positive cells, the neurological functional deficits and the brain infarct volume. These results suggest that the SUR1-regulated NCCa-ATP channel may be associated with MCAO/reperfusion injury and the infarct-reducing effects of intravenous treatment with gliclazide may be due, in part, to the blocked upregulation of SUR1 expression, the decreased infarct size and the reduced apoptosis in the ischemia–reperfusion brain.

Expression of netrin-1 and its receptors, deleted in colorectal cancer and uncoordinated locomotion-5 homolog B, in rat brain following focal cerebral ischemia reperfusion injury.

Netrin-1 is currently one of the most highly studied axon guidance factors. Netrin-1 is widely expressed in the embryonic central nervous system, and together with the deleted in colorectal cancer and uncoordinated locomotion-5 homolog B receptors, netrin-1 plays a guiding role in the construction of neural conduction pathways and the directional migration of neuronal cells. In this study, we established a rat middle cerebral artery ischemia reperfusion model using the intraluminal thread technique. Immunofluorescence microscopy showed that the expression of netrin-1 and deleted in colorectal cancer in the ischemic penumbra was upregulated at 1 day after reperfusion, reached a peak at 14 days, and decreased at 21 days. There was no obvious change in the expression of uncoordinated locomotion-5 homolog B during this time period. Double immunofluorescence labeling revealed that netrin-1 was expressed in neuronal cells and around small vessels, but not in astrocytes and microglia, while deleted in colorectal cancer was localized in the cell membranes and protrusions of neurons and astrocytes. Our experimental findings indicate that netrin-1 may be involved in post-ischemic repair and neuronal protection via deleted in colorectal cancer receptors.

Endogenous tissue plasminogen activator increases hemorrhagic transformation induced by heparin after ischemia reperfusion in rat brains

Objective: Tissue plasminogen activator (tPA) as a main thrombolytic drug for acute ischemic stroke remains complicated by risk of hemorrhagic transformation. However, whether endogenous tPA is also involved in hemorrhagic transformation is yet unclear.Methods: We randomly assigned male Sprague–Dawley rats into three groups: the heparin group, the control group and the sham operated group. The ischemic rat models were induced by middle cerebral artery occlusion through intraluminal thread technique for 2 hours, followed by 24 hours of reperfusion. Heparin or saline was intermittent peritoneally injected after reperfusion. The extent of cerebral hemorrhage, the infarct volume, as well as the content and activity of endogenous tPA were evaluated. The matrix metalloproteinase 9 (MMP-9) expression and activity were also measured.Results: All rats receiving heparin after reperfusion were subjected to hemorrhagic transformation. Hemorrhage volume in the heparin group was remarkably present. There was significant difference between the two groups (p<0·01). In the heparin group, the expressions of endogenous tPA and MMP-9 obviously increased, while their content and activity had significant differences compared with that of the control group (p<0·01).Conclusion: Endogenous tPA, through enhancement of MMP-9 expression and proteolytic activation, plays an important role in the pathogenesis of hemorrhagic transformation after cerebral reperfusion induced by heparin.

Dynamic changes of the expression of Notch(1) and Jagged(1) in the hippocampus after focal ischemia: experiment with rats

To detect the expression patterns of Notch(1) and Jagged(1) in the hippocampus of focal ischemia and find out whether the Notch pathway is involved in the regulation of the differentiation of the proliferated neural precursor cells in the dentate gyrus under ischemic conditions. 42 SD rats were randomly divided into 3 equal groups: middle cerebral artery occlusion (MCAO) group undergoing MCAO by intraluminal thread technique so as to cause focal cerebral infarction, sham operation group, and normal control group.19 and 28 days after the operation 6 rats in each group were killed with their hippocampus taken out. The protein and mRNA levels of Notch(1) and Jagged(1) in hippocampus were assayed respectively by Western blotting and real-time reverse-transcription PCR. The protein and mRNA expression levels of Notch(1) on the 19th day of the MCAO group were up-regulated, both significantly higher than those of the sham operation and control groups (all P < 0.05). The protein and mRNA expression levels of Notch(1) on the 28th day of the MCAO group were both significantly lower than those of the same group on the 19th day (both P < 0.01), and were lower than those of the sham operation and control groups on the 28th day, however, not significantly (all P > 0.05). The protein and mRNA expression levels of Jagged(1) on the 19th and 28th days of the MCAO group were significantly higher than those of the sham and control groups (all P < 0.05), however, there were not significant differences in the protein and mRNA expression levels of Jagged(1) on the 19th and 28th days of the MCAO group (all P > 0.05). The protein and gene expression of Notch(1) and Jagged(1) vary dynamically in the hippocampus of focal ischemia, which suggests that the Notch pathway may be a potential mechanism in the regulation of the neurogenesis in the dentate gyrus of hippocampus.

Brain damage related to hemorrhagic transformation following cerebral ischemia and the role of KATP channels

Background: Hemorrhagic transformation (HT) is a major factor limiting the use of thrombolytic treatment for stroke. Animal model can help us to understand HT. This study is to establish a HT model in rats to compare HT with uncomplicated cerebral infarction in neurobehavioral deficit, brain edema, brain adenosine triphosphatase (ATPase) activity and succinic dehydrogenase (SDH) activity, and to investigate its pathology changes as well as the impact of, Glibenclamide, a ATP-sensitive K+ channel (KATP channel) blocker, on the pathogenesis of HT. Methods: Male, Sprague–Dawley rats were randomly assigned to four groups: hemorrhagic transformation (HT), cerebral infarction (CI), Glibenclamide+HT (GH) and a control. To create HT model, right middle cerebral artery occlusion (MCAO) was conducted with intraluminal thread technique; 30 min after MCAO, 50 μL arterial blood was injected into the caudate nucleus where the infarction occurred. Neurologic deficit was evaluated by Longa test, Berderson test and Beam test. Brain water content, brain ATPase activity and SDH activity were measured. Histology was examined using light microscope and transmission electron microscope. Results: No significant difference in neurobehavioral deficit and brain water content was observed between HT and CI groups in all time points (P>0.05). Brain ATPase activity 12 h and 24 h after operation and brain SDH activity 24 h after the operation in HT group were both significantly increased compared with those in CI group (P<0.05); the increase of brain ATPase and SDH activity in HT group could be prevented by Glibenclamide. Neuronal degeneration and tissue edema in HT group, swollen neuropil and loosen intercellular substance in CI group were revealed by histology study. Ultrastructural changes including swollen mitochondria and interstitial edema were also observed in both HT and CI groups. Conclusions: The results demonstrated that moderate hemorrhagic transformation does not significantly aggravate cerebral infarction, and that KATP channels have an important role in energy metabolism.

Chronic intracerebroventricular delivery of the secretory phospholipase A2 inhibitor, 12-epi-scalaradial, does not improve outcome after focal cerebral ischemia–reperfusion in rats

Phospholipase A2s (PLA2s) seem to be involved in the pathophysiology of ischemic brain injury, but their specific role is far from being completely understood. The present study was carried out to ascertain how and to what extent secretory PLA2s (sPLA2s) activity influences outcome after cerebral ischemia-reperfusion, and to correlate this with the inflammatory response. To do this we used the potent and selective sPLA2 inhibitor, 12-epi-scalaradial. Male Wistar rats were separated into three groups: a control group receiving intracerebroventricular vehicle, and two groups receiving intracerebroventricular 0.005 or 0.5 microg/h 12-epi-scalaradial. Every animal was subjected to middle cerebral artery (MCA) occlusion (90 min, intraluminal thread technique) under continuous moni-torization of cerebrocortical perfusion (CP, laser-Doppler flowmetry), followed by reperfusion (3 days). Neurological status, infarct volume, and myeloperoxidase (MPO) activity were the main end points. Three days after the 90-min ischemia period, neurological examination did not reveal significant differences between the three groups of rats. Control rats showed a mean infarct volume of 145.9 +/- 24.7 mm3 (21 +/- 4.1% of the ipsilateral hemisphere volume), while mean infarct volume in rats treated with 0.005 or 0.5 microg/h 12-epi-scalaradial increased to 164.8 +/- 86.8 mm3 (22.0 +/- 10.9%) and 211.5 +/- 12.2 mm3 (28 +/- 3%, P < 0.05), respectively. Treatment with the highest dose of 12-epi-scalaradial (0.5 microg/h) increased MPO activity in the ipsilateral hemisphere by about 140% (from 0.59 +/- 0.59 to 1.42 +/- 1.03 units of activity/g of tissue in comparison with the control ischemic hemisphere, P < 0.05). Overall, our results point to a positive rather than a negative influence of sPLA2 activity during ischemia. This, along with its inability to decrease the inflammatory response, does not allow to propose the use of 12-epi-scalardial as a potential drug for stroke therapy.

Dietary phytoestrogens improve stroke outcome after transient focal cerebral ischemia in rats

As phytoestrogens are postulated as being neuroprotectants, we assessed the hypothesis that dietary isoflavone-type phytoestrogens are neuroprotective against ischemic stroke. Transient focal cerebral ischemia (90 min) was induced by middle cerebral artery occlusion (MCAO) following the intraluminal thread technique, both in rats fed with soy-based diet and in rats fed with isoflavone-free diet. Cerebro-cortical laser-Doppler flow (cortical perfusion, CP), arterial blood pressure, core temperature, PaO2, PaCO2, pH and glycemia were measured before, during and after MCAO. Neurological examination and infarct volume measurements were carried out 3 days after the ischemic insult. Dietary isoflavones (both glycosides and aglycones) were measured by high-performance liquid chromatography. Neither pre-ischemic, intra-ischemic nor post-ischemic CP values were significantly different between the soy-based diet and the isoflavone-free diet groups. Animals fed with the soy-based diet showed an infarct volume of 122 +/- 20.2 mm3 (19 +/- 3.3% of the whole ipsilateral hemisphere volume). In animals fed with the isoflavone-free diet the mean infarct volume was significantly higher, 191 +/- 26.7 mm3 (28 +/- 4.1%, P < 0.05). Neurological examination revealed significantly higher impairment in the isoflavone-free diet group compared with the soy-based diet group (3.3 +/- 0.5 vs. 1.9 +/- 0.5, P < 0.05). These results demonstrate that dietary isoflavones improve stroke outcome after transient focal cerebral ischemia in such a way that a higher dietary isoflavone content results in a lower infarct volume and a better neurological status.

Transfemoral selective “intraluminal wiring” technique for transient middle cerebral artery occlusion in rats

While the intraluminal thread technique to induce middle cerebral artery occlusion is widely used in animal models of focal cerebral ischemia, it has several drawbacks. The present study describes a new technique involving transfemoral selective intraluminal wiring, and evaluates its technical feasibility, effectiveness, and safety. Twenty-four Wistar rats were used in this work: two for a vascular anatomy study and 22 subjected to middle cerebral artery occlusion for 1 h by our new transfemoral selective “intraluminal wiring” technique. After 24 h of reperfusion, the animals were evaluated neurologically, and then were sacrificed. Macroscopic, histological (2,3,5-triphenyltetrazolium chloride (TTC), hematoxylin–eosin and TUNEL), and biochemical (DNA fragmentation and caspase-3 activity) studies were performed to assess the extent of brain damage produced by focal ischemia. Technical success was obtained in all 22 animals. Signs of focal ischemia and reperfusion, such as necrosis and apoptosis, were detected in the middle cerebral artery territory. No subarachnoid hemorrhage was noticed in any animal. Transfemoral selective intraluminal wiring appears to be a reliable, safe, and minimally invasive technique to induce transient focal cerebral ischemia in rats.

Inhibition of iNOS activity by 1400W decreases glutamate release and ameliorates stroke outcome after experimental ischemia

It has been shown that the reversed operation of glutamate transporters when ATP levels fall accounts for most glutamate release induced by severe cerebral ischemia. Nitric oxide (NO) is formed after ischemia and causes ATP depletion. Our purpose is to test if NO release from inducible NO synthase (iNOS) after stroke may cause a delayed glutamate release due to ATP depletion that might underlie progression of the ischemic infarct. We have studied the effect of the highly selective inhibitor of iNOS activity 1400W on brain ATP levels, extracellular glutamate, and stroke outcome after transient focal cerebral ischemia in rats. To induce focal ischemia, the middle cerebral artery (MCA) was occluded by using the intraluminal thread technique (tMCAO). 1400W was administered, after tMCAO, by using an Alzet osmotic pump to yield a drug delivery rate of 2.5 mg/kg/h. Results. Postischemic treatment with 1400W induced a reduction in the neurofunctional impairment and in the total volume of brain infarct. Western blot analysis showed ischemia-induced expression of iNOS. Treatment with 1400W partially prevented delayed ATP reduction and produced inhibition of the subsequent delayed increase in glutamate levels caused by the ischemic insult. Our data indicate that 1400W improves stroke outcome, an effect concomitant to the inhibition of both ischemia-induced decrease in brain ATP levels and increase in glutamate release. These results provide evidence indicating that the expression of iNOS induced by ischemia may contribute to the progression of the ischemic infarct and have important therapeutic implications for the management of stroke.

Improvements in the intraluminal thread technique to induce focal cerebral ischaemia in rabbits

Although the intraluminal thread technique has been used to induce focal cerebral ischaemia in rabbits, its success rate is not high. We, therefore, attempted to improve the stability and reproducibility of this method by using thread tips of appropriate diameter as determined from the anatomical characteristics of the carotid and cerebral arteries of New Zealand white rabbits. Following intraarterial injection of casting material, we tested threads of four different tip diameters to determine the optimal thread tip that could occlude the middle cerebral artery (MCA). 2,3,5-Triphenyltertrazolium chloride (TTC) staining showed that, consistent with the intraluminal diameter of the MCA from the arterial casts (0.50±0.06 mm), thread with 0.51–0.55 mm tip diameter was optimal for the occlusion of the MCA. Ability to induce focal cerebral ischaemia was also dependent on variations in the anatomy of the internal carotid artery (ICA), especially the origin of the occipital artery. Our results suggest that use of appropriately sized thread and accurate manipulation of its tip significantly improves the stability and reproducibility of this model.

Improvements in the intraluminal thread technique to induce focal cerebral ischaemia in rabbits

Although the intraluminal thread technique has been used to induce focal cerebral ischaemia in rabbits, its success rate is not high. We, therefore, attempted to improve the stability and reproducibility of this method by using thread tips of appropriate diameter as determined from the anatomical characteristics of the carotid and cerebral arteries of New Zealand white rabbits. Following intraarterial injection of casting material, we tested threads of four different tip diameters to determine the optimal thread tip that could occlude the middle cerebral artery (MCA). 2,3,5-Triphenyltertrazolium chloride (TTC) staining showed that, consistent with the intraluminal diameter of the MCA from the arterial casts (0.50±0.06 mm), thread with 0.51–0.55 mm tip diameter was optimal for the occlusion of the MCA. Ability to induce focal cerebral ischaemia was also dependent on variations in the anatomy of the internal carotid artery (ICA), especially the origin of the occipital artery. Our results suggest that use of appropriately sized thread and accurate manipulation of its tip significantly improves the stability and reproducibility of this model.

Temporal changes in sensitivity of rats to cerebral ischemic insult.

Experimental rat models are often used to study cerebral ischemia, yet rats are nocturnal animals that have activity cycles that are the opposite of those of humans. In the following study the authors examined the circadian rhythm of sensitivity to an ischemic insult in rats by using an intraluminal thread technique to produce reversible middle cerebral artery occlusion. Ischemia (2 hours of blockage followed by 22 hours of reperfusion) was induced in rats according to the 24-hour clock at either 100, 400, 700, 1,000, 1,300, 1,600, 1,900, or 2,200 hours (11-14 rats per time period). The rat brains were removed, coronally sectioned, stained with 2,3,5-triphenyltetrazolium chloride and analyzed using commercially available software. Analysis of variance and cosinor-rhythmometry statistical tests were used for analysis of data. The time of day when the ischemic infarct was induced had a significant (p = 0.011) influence on the volume of the lesion. The volume of total brain infarct produced at 400 hours (7.65 +/- 1.31%) was more than three times greater than the volume produced at 1600 hours (2.1 +/- 0.34%). Cosinor-rhythm analysis indicated a peak occurrence of infarct volume at 6:02 (95% confidence interval 5:49-6:16). The size of the infarct correlated with core body temperature rhythms, which varied by 1.3 +/- 0.62 degrees C (mean +/- standard deviation). Circadian rhythms, as well as the reversed natural body rhythms of the rat compared with humans, should be considered when extrapolating data to human or other animal studies. Temporal rhythms may also provide information concerning the cascading disease processes associated with cerebral ischemia.

A temporal MRI assessment of neuropathology after transient middle cerebral artery occlusion in the rat: correlations with behavior.

The purpose of this study was to evaluate the temporal and spatial pathological alterations within ischemic tissue using serial magnetic resonance imaging (MRI) and to determine the extent and duration of functional impairment using objective behavioral tests after transient middle cerebral artery occlusion (tMCAO) in the rat. MRI signatures derived from specific anatomical regions of interest (ROI) were then appropriately correlated to the behavioral measures over the time course of the study (up to 28 days post-tMCAO). Sprague-Dawley rats (n = 12) were initially trained on the following behavioral tasks before surgery: bilateral sticky label test (for contralateral neglect); beam walking (for hindlimb coordination); staircase test (for skilled forelimb paw-reaching). Rats were then randomly assigned to receive either tMCAO (90 minutes, n = 6), by means of the intraluminal thread technique, or sham-control surgery (n = 6). Proton density, T2- and T2-diffusion-weighted MR images were acquired at 1, 7, 14, and 28 days post-tMCAO that were then smoothed into respective proton density, T2 relaxation, and apparent diffusion coefficient (ADC) maps. Apparent percent total lesion volume was assessed using T2W imaging. MR signatures were evaluated using the tissue maps by defining ROI for MCAO and sham-control groups, which corresponded to the caudate-putamen, forelimb, hindlimb, and lower parietal cortices both ipsilateral and contralateral to the occlusion site. Behavioral tests were undertaken daily from 1 to 28 days post-tMCAO. Results demonstrate that apparent percent lesion volume reduced from 1 to 7 days (P < 0.05) but then remained constant up to 28 days for the MCAO group. Pathological changes in the temporal profile of T2 and ADC tissue signatures were significantly altered in specific ROI across the time course of the study (P < 0.05 to <0.001), reflecting the progression of edema to necrosis and cavitation. Both T2 and ADC measures of ischemic pathology correlated with parameters defined by each of the functional tests (r > or =0.5, P < 0.05) across the time course. The staircase test revealed bilateral impairments for the MCAO group (P <0.001), which were best predicted by damage to the ipsilateral lower parietal cortex by means of hierarchical multiple regression analyses (R2 changes > or =0.21, P < or =0.03). Behavioral recovery was apparent on the beam walking test at 14 to 28 days post-MCAO, which was mirrored by MRI signatures within the hindlimb cortex returning to sham-control levels. This long-term study is the first of its kind in tracing the dynamic pathologic and functional consequences of tMCAO in the rat. Both serial MRI and objective behavioral assessment provide highly suitable outcome measures that can be effectively used to evaluate promising new antiischemic agents targeted for the clinic.

Interrupted Arterial Occlusion Reduces Ischemic Damage in a Focal Cerebral Ischemia Model of Rats

It is generally thought by neurosurgeons that when temporary clipping of a major cerebral vessel is necessary during aneurysm surgery, repeated short periods of cerebral ischemia are safer for the brain than a single long episode. This study was performed to investigate whether repetitive short episodes of cerebral ischemia would alter the resulting brain injury as compared with a single long period of ischemia in a rat model for focal cerebral ischemia. Middle cerebral artery occlusion and reperfusion were performed by the intraluminal thread technique. The experimental design consisted of a single 90-minute occlusion period in the continuous ischemia group versus three 30-minute occlusion periods with 15-minute reperfusion periods in the repetitive group. Local cerebral blood flow was measured by the hydrogen clearance technique. During the ischemic period, local cerebral blood flow values significantly decreased in both the continuous and the repetitive groups. Cerebral blood flow restoration was demonstrated after each episode of reperfusion in both groups. The neurological status scores 2 hours after surgery in the rats subjected to repetitive insults were significantly better compared with those in the rats of the continuous ischemia group. However, the scores on Days 1, 3, and 7 did not show a significantly better difference. The animals were killed 7 days after the induction of ischemia for the measurement of the infarction area under the microscope. The total area of infarction was significantly reduced (4.05 +/- 4.56 versus 47.2 +/- 37.3 mm2, P < 0.001) by interruption of the ischemic time period.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversible middle cerebral artery occlusion in rats using an intraluminal thread technique

We investigated the temporal profile for neuropathologic outcomes after cerebral ischemia using a rat model of reversible middle cerebral artery occlusion, where reperfusion can be introduced in nonanesthetized rats. Reperfusion was performed 1 hour to 5 hours after the occlusion. Control animals underwent permanent occlusion. The results indicate that the time window to reduce infarct volume is 2 hours, and that a ≥3-hour duration of ischemia is sufficient to attain the maximal infarction observed after permanent ischemia. This suggests that any therapies that follow the therapeutic window will provide little benefit for transient cerebral ischemia and reperfusion injury.