Brain cells manufacture and secrete angiogenic peptides after focal cerebral ischemia, but the purpose of this angiogenic response is unknown. Because the maximum possible regional cerebral blood flow is determined by the quantity of microvessels in each unit volume, it is possible that angiogenic peptides are secreted to generate new collateral channels; other possibilities include neuroprotection, recovery/regeneration, and removal of necrotic debris. If the brain attempts to create new collaterals, microvessel density should increase significantly after ischemia. Conversely, if angiogenic-signaling molecules serve some other purpose, microvessel densities may increase slightly or not at all. To clarify, the authors measured microvessel densities with quantitative morphometry. Left middle cerebral arteries of adult male Sprague–Dawley rats were occluded with intraluminal nylon suture for 4 hours followed by 7, 14, 19, or 30 days of reperfusion. Controls received no surgery or suture occlusion. Changes in microvessel density and macrophage numbers were measured by light microscopic morphometry using semiautomated stereologic methods. Microvessel density increased only in the ischemic margin adjacent to areas of pannecrosis and was always associated with increased numbers of macrophages. Ischemic brain areas without macrophages displayed no vascularity changes compared with normal animals. These data suggest that ischemia-induced microvessels are formed to facilitate macrophage infiltration and removal of necrotic brain.

Previous methods for determining morphological features of vascular networks in cerebral cortex were subject to arbitrary variation and bias. Unbiased estimates of vessel number, volume, surface area and length can be obtained using stereology but these techniques tend to be tedious and time-consuming. Stereologic protocols generally require micrographs that have to be analyzed manually for intersections of vessels on grid points or lines. In this report, we provide a simpler and more precise method for measuring morphological features of cerebral cortical microvessels. Images of microvessels in 1 μm toluidine blue stained sections were captured using a popular image analysis software package. Luminal surfaces of endothelial cells were automatically traced using commonly available features; the two-dimensional data of vessels (diameter, area, perimeter and number of vessels) were automatically computed and transferred to a spreadsheet. Three-dimensional features were then determined using basic stereologic equations. The method eliminates the need for manual measurements and is particularly time- and cost-effective for quantitative studies where numerous images have to be evaluated.

We sought to prolong the window for stroke treatment using synergistic combinatorial therapy. We used the intraluminal filament occlusion model in rats to cause focal cerebral ischemia and a quantal bioassay to measure efficacy. The GABA agonist muscimol and the glutamate antagonist MK-801 were used alone and in combination at various times after ischemia onset. At progressively longer treatment delay intervals (30, 60, 75, 120, 240, and 360 min), higher doses of the single drugs were required to achieve neuroprotection. In contrast, the combination 1.0 mg/kg muscimol plus 0.5 mg/kg MK-801 was effective at all delay intervals studied except the longest (P < 0.05 at each time). After 240 min from ischemia onset, the combination was more effective than either single agent (P < 0.05 for each drug dose), suggesting synergism. The neuroprotective effect could not be demonstrated using morphometry. The treatment effects were probably not due to hypothermia because brain temperatures recorded in awake, unregulated subjects remained normo- or slightly hyperthermic following all treatments. Awake subjects kept on a heating pad exhibited mild brain hyperthermia. The combination caused a drop and MK-801 caused a significant increase in mean arterial blood pressure (main effects F(5,172) = 29, P < 0.0001). The combination of a GABA agonist and glutamate antagonist appears to possess synergistic neuroprotective effects when treatment is delayed up to 240 min following the onset of cerebral ischemia. Temperature regulation causes hyperthermia in awake subjects. The quantal bioassay is one method suitable for studies of synergistic stroke therapy.

OBJECTIVE:To evaluate the effect of hyperbaric oxygen (HBO) therapy on neurologic outcome (assessed by the quantal bioassay approach) following acute focal cerebral ischemia in rats.METHODS:Two separate experimental trials were conducted. Trial 1, a nonblinded experiment, used 38 rats. Trial 2, a blinded experiment, used 59 rats. Focal cerebral ischemia was induced with a surgically placed intraluminal occlusion of the left middle cerebral artery; subsequent removal allowed reperfusion. Arterial occlusion times were varied from 5 to 90 minutes in trial 1, and from 3 to 45 minutes in trial 2. The control groups were maintained at ambient pressure for the duration of each trial. The trial 1 treatment group received a single 30-minute HBO treatment at 2.0 atmospheres absolute (ATA) on the initial day of ischemia. The trial 2 treatment group received 30-minute HBO treatments at 2.0 ATA daily for 4 consecutive days. The animals underwent daily 5-point neurologic examinations. A computerized quantal bioassay was used to determine the ET50--the occlusion time required to cause a neurologic abnormality in half of the animals. The control and treatment ET50 values were compared in each trial using a 2-tailed t-test. An increased ET50 for the treatment vs the control group implied a beneficial effect of HBO; a decrease, the opposite. The study had a power of 80% to detect a difference of 11.4 minutes in the ET50 for a 2-sided alpha = 0.05.RESULTS:For trial 1: the HBO ET50 was 18.1 +/- 21.9 minutes and the control ET50 was 22.8 +/- 25.0 minutes (p > 0.2). For trial 2: the HBO ET50 was 9.49 +/- 17.4 minutes and the control ET50 was 14.9 +/- 14.2 minutes (p < 0.2).CONCLUSION:HBO therapy showed no apparent benefit in a rat model as a treatment modality for acute cerebral ischemia with reperfusion.

Agonists of the GABA-A receptor are neuroprotective after experimental stroke, but studies of GABA-B agonists have contradicted each other. To further investigate whether GABA-B agonists may be neuroprotective, we devised a quantal bioassay using the intraluminal occlusion method of inducing reversible cerebral ischemia. Subjects underwent middle cerebral artery occlusion for varying amounts of time, ranging from 5 to 90 min. Behavioral outcome was measured 48 h later with a quantal observational scale: score of abnormal given for any one of asymmetric forepaw flexion on tail lift, asymmetric grip, circling, reduced exploration, seizures, or death. To the grouped response data the logistic equation was used to find the ED50, the duration of occlusion that caused one-half of the subjects to be abnormal. To find the potency ratio for each drug, we divided the ED50for treatment by that for vehicle. We administered baclofen, a GABA-B agonist, intraperitoneally 5 min after the onset of ischemia. Baclofen (20 mg/kg) was neuroprotective (potency ratio of 3.0,P < 0.05), but a lower dose (10 mg/kg) was not. However, both doses of baclofen caused significantly more intracerebral hemorrhages than control. In awake animals, both baclofen doses caused significant increases in mean arterial pressure, but no changes in other cardiorespiratory variables. The glutamate antagonist MK-801, the GABA-A agonist muscimol, and hypothermia were all protective using the bioassay (potency ratios ranging from 1.5 to 3.0). We conclude that although baclofen (20 mg/kg) may be neuroprotective, its utility is complicated by postischemic hypertension and cerebral hemorrhages.