James McCulloch

Focal Cerebral Ischaemia in the Rat: 1. Description of Technique and Early Neuropathological Consequences Following Middle Cerebral Artery Occlusion

A procedure for occluding the stem of the proximal middle cerebral artery of the rat is described. The operation is performed under anaesthesia through a small subtemporal craniectomy. After occlusion, 3 animals were perfused with carbon black and 8 with a FAM fixative (40% formaldehyde, glacial acetic acid, and methanol). The findings were compared with sham-operated animals. Carbon black studies demonstrated an area of impaired perfusion corresponding to the territory of the occluded artery in each animal. Neuropathological studies invariably showed that there was ischaemic brain damage in the cortex and basal ganglia. The frontal cortex was involved in every animal, as was the lateral part of the neostriatum; the sensorimotor and auditory cortex were involved in most animals, whereas the occipital cortex and medial striatum were involved only infrequently. The damage produced by ischaemia could be readily distinguished from the small local lesion seen at the surgical site in sham-operated animals. The ability to produce a consistent focal ischaemic lesion in the rodent brain provides a technical approach that is sufficiently reproducible to enable investigation of the pathophysiology of ischaemia using recently developed autoradiographic and neurochemical methods.

Protective Effect of the Glutamate Antagonist, MK-801 in Focal Cerebral Ischemia in the Cat

The effects of the glutamate N-methyl-D aspartate (NMDA) receptor antagonist, MK-801, upon ischemic brain damage has been examined in anesthetized cats. Focal cerebral ischemia was produced by permanent occlusion of one middle cerebral artery and the animals were killed 6 h later. The amount of early ischemic damage was assessed in coronal sections at 16 predetermined stereotactic planes. Pretreatment with MK-801 (5 mg/kg, i.v.), 30 min before occlusion of the middle cerebral artery significantly reduced the volume of ischemic damage (from 32.7 ± 4.0% of the cerebral hemisphere in vehicle-treated cats to 16.2 ± 4.5% in MK-801-treated cats). NMDA receptor antagonists that penetrate the blood-brain barrier, such as MK-801, merit further study as protective agents against ischemic brain damage.

Innervation of the feline cerebral vasculature by nerve fibers containing calcitonin gene-related peptide: Trigeminal origin and co-existence with substance P

The presence of a population of nerve fibers containing immunoreactive calcitonin gene-related peptide (CGRP) has been demonstrated around cerebral arteries of the cat with immunocytochemistry and radioimmunoassay. CGRP immunoreactivity in the feline cerebral vasculature, as characterized by high-performance liquid chromatography, is similar to authentic rat CGRP. Numerous perikarya containing CGRP are present in the trigeminal ganglia, and surgical lesions of the trigeminal ganglia significantly reduce the levels of CGRP in the cerebral vasculature, suggesting that this cranial nerve is the principal origin of these cerebrovascular nerve fibers. As demonstrated by sequential immunocytochemistry, CGRP coexists with substance P both in the trigeminal ganglion and nerve fibers around cerebral blood vessels. The presence of CGRP in the cerebrovascular trigeminal innervation provides further versatility and complexity for this sensory afferent system putatively involved in the transmission of intracranial pain.

Neuropeptide Y: Cerebrovascular innervation an vasomotor effects in the cat ☆

Avian pancreatic polypeptide (APP) has been proposed to be a neurotransmitter in a subpopulation of sympathetic nerves. Here, we present immunocytochemical and pharmacological evidence that the structurally related peptide, neuropeptide Y (NPY), is likely to be the biologically active material in these nerves. Cerebral arteries from cats are invested with a dense network of NPY-containing nerve fibres, as demonstrated by immunocytochemistry. This immunoreaction is abolished by prior removal of the superior cervical ganglion. NPY causes strong contractions of cerebral arteries in vitro whereas APP has small effects on the vasomotor reactivity. The NPY-induced contractions were not inhibited by the alpha 2-adrenoceptor antagonist rauwolscine (10(-7) M) or the 5-hydroxytryptamine antagonist ketanserin (10(-7) M). The contractions were, however, sensitive to calcium removal or to the calcium antagonist diltiazem (10(-4) M).

Quantitative assessment of early brain damage in a rat model of focal cerebral ischaemia.

A method for the volumetric assessment of early cerebral infarction, together with its statistical and biological validation, is described. In halothane anaesthetised rats the stem of the right middle cerebral artery was occluded and 3 hours later (with full monitoring of respiratory and cardiovascular status) the animals were killed by perfusion fixation. In normotensive normocapnic animals the volume of infarction was 52 +/- 4 mm3 in the cerebral cortex and 21 +/- 1 mm3 in the corpus striatum. The reproducibility of the volumetric assessment was found to be excellent (coefficient of correlation 0.995 on 18 replicate measurements). The minimum number of stereotactic levels which must be assessed to yield accurate volumetric measurements of infarction is 8. The method is sensitive at detecting alterations in the amount of infarction. For example, it can readily detect the increase in amount of structural damage in cerebral cortex following a transient episode of hypotension. This approach allows an objective assessment of drug therapy and management strategies in the treatment of cerebral infarction.

Focal Cerebral Ischaemia in the Rat: 2. Regional Cerebral Blood Flow Determined by [14C]Iodoantipyrine Autoradiography following Middle Cerebral Artery Occlusion

Local cerebral blood flow has been measured by quantitative autoradiography, employing [14C]iodoantipyrine as tracer, in rats killed half an hour after occlusion of the middle cerebral artery. The results were compared with pattern of local cerebral blood flow (CBF) in sham-operated rats and with neuropathological findings. In every animal there was a profound reduction (to 13% of control levels) in blood flow in the neocortex previously supplied by the occluded artery. The level of blood flow in the areas in which ischaemic brain damage occurred was 0.24 ±0.03 ml g−1 min−1 (mean ± SEM). This level of CBF is considerably greater than that reported following a similar surgical procedure in cats and primates. Moderate reductions in blood flow were also seen outside the territory of the occluded artery and in parts of the opposite hemisphere. Absolute increases in blood flow (hyperaemia) were seen only in the substantia nigra and globus pallidus ipsilateral to the occlusion. It is suggested that this finding and the reductions in blood flow outside the territory of the middle cerebral artery are reflections of alterations in neuronal function and metabolic activity secondary to the ischaemic lesion.

Calcitonin Gene-Related Peptide and Cerebral Blood Vessels: Distribution and Vasomotor Effects

The innervation of cerebral blood vessels by nerve fibers containing calcitonin gene-related peptide (CGRP) and the vasomotor effects of this peptide are described for a number of different mammalian species. CGRP-immunoreactive nerve fibers were present in the adventitia of cerebral arteries in all species examined (guinea pig, cat, rabbit, rat, and mouse). Numerous perikarya containing CGRP immunoreactivity are demonstrable in the trigeminal ganglion of all species. In the cerebral perivascular nerve fibers and in trigeminal perikarya, CGRP is often colocalized with substance P and neurokinin A. Marked interspecies differences exist both in the density of CGRP-immunoreactive nerve fibers and in the cerebrovascular levels measured with radioimmunoassay. The highest concentrations were observed in cerebral vessels from guinea pigs, the lowest concentration in rabbit vessels, and intermediate levels in the feline and human cerebral vasculature. CGRP is a potent dilator of cerebral arteries in all species examined (human pial, feline middle cerebral, rabbit, guinea pig and rat basilar arteries). The concentration of CGRP eliciting half-maximal responses ranged from 0.4 nM (human pial artery) to 3 nM (rat and rabbit basilar arteries). Pretreatment of cerebral arteries with low concentrations of either substance P (0.1 nM) or neurokinin A (3 nM) attenuated slightly the CGRP-induced relaxations of guinea pig basilar arteries. Calcitonin was found to be a very weak dilator of cerebral arteries from human and guinea pig. Thus, cardiovascular nerve fibers containing CGRP appear to be present in all mammalian species (although to varying degrees) and CGRP is invariably a potent dilator of the cerebral arteries for all species.

Focal Cerebral Ischaemia in the Cat: Treatment with the Glutamate Antagonist MK-801 After Induction of Ischaemia

The effects of the glutamate N-methyl-D-aspartate receptor antagonist MK-801 in reducing ischaemic brain damage have been examined in anaesthetised cats, with drug treatment being initiated 2 h after the induction of cerebral ischaemia. Focal cerebral ischaemia was produced by permanent occlusion of one middle cerebral artery, and the animals were killed 6 h later. The amount of early irreversible ischaemic damage was assessed at 16 predetermined stereotactic planes. Treatment with MK-801 (5 mg/kg, i.v.) 2 h after middle cerebral artery occlusion reduced significantly the volume of ischaemic damage (from 1,625 ± 384 mm3 of the cerebral hemisphere in vehicle-treated cats to 792 ± 385 mm3 in MK-801-treated cats). The demonstration of reduced ischaemic brain damage with MK-801, when the agent is administered after the induction of ischaemia, extends the therapeutic potential of such agents in the treatment of focal cerebral ischaemia in humans.

Endothelin-1-Induced Reductions in Cerebral Blood Flow: Dose Dependency, Time Course, and Neuropathological Consequences

The capacity of endothelin-1 to induce severe reductions in cerebral blood flow and ischaemic neuronal damage was assessed in anaesthetised rats. Endothelin-1 (25 μl of 10−7–10−4 M) was applied to the adventitial surface of an exposed middle cerebral artery and striatal blood flow assessed by the hydrogen clearance technique. Endothelin-1 induced severe dose-dependent reductions in cerebral blood flow (e.g., minimum CBF at 10−5 M of 9 ± 11 ml 100 g−1 min−1 compared to 104 ± 22 ml 100 g−1 min−1 with vehicle, p < 0.05), which persisted for at least 60 min at each concentration of endothelin-1. Application of endothelin-1 to the middle cerebral artery produced dose-dependent ischaemic brain damage (e.g., volume of damage of 65 ± 34 mm3 at 10−5 M compared to 0.22 ± 0.57 mm3 for vehicle, p < 0.01). These data demonstrate that endothelin-1 is capable of reducing blood flow to pathologically low levels and provide a new model of controlled focal ischaemia followed by reperfusion.

Substance P: immunohistochemical localization and effect upon cat pial arteries in vitro and in situ.

1. Nerve fibres containing substance P immunoreactivity were present in the adventitia and the adventitia‐media border of all cat cerebral arteries which were examined. Substance P immunoreactivity was most abundant in cerebral arteries from the rostral portion of the circle of Willis. 2. Substance P effected a dose‐dependent relaxation of feline middle cerebral arteries which had been contracted with prostaglandin F2 alpha. The maximum relaxation (16 +/‐ 0.3 mN) was achieved with substance P at a concentration of 10‐6 M. 3. In cats anaesthetized with alpha‐chloralose, the perivascular microinjection of substance P effected dose‐dependent increases in arteriolar calibre. The maximum increase in calibre (19 +/‐ 3%) was observed following the injection of 10(‐6) M‐substance P.