SUMMARY 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 + 4mm3 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 0995 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.Numerous attempts have been made to produce experimentally an ischaemic lesion resembling that seen in man. One of the most commonly used methods has been to occlude the middle cerebral artery in various experimental animals`6 in the belief that proximal occlusion of this artery is equivalent to a large focal cerebral infarct in man.We have developed a technique for occluding the middle cerebral artery (MCA) in the rat7 which has proved useful for correlating local cerebral blood flow8 and dysfunction of the blood-brain barrier9 with histological evidence of ischaemic brain damage. 10 In this paper we detail a rigorous means for measuring the volume of early cerebral infarction in the rat after occlusion of the middle cerebral artery. This method, with its statistical validation, together with a demonstration of its ability to measure changes in the amount of structural damage in various experimental conditions, is described. Some of this data has appeared previously in abstract form." Materials and methods General preparationThirteen adult male Sprague Dawley rats (weighing between 320-415 g each) were anaesthetised and ventilated mechanically with a nitrous oxide/oxygen mixture (70%:30%) and, during the surgical procedures, 2% halothane. Polyethylene catheters were inserted into both femoral arteries and veins to allow the continuous monitoring of blood pressure, repeated sampling of arterial blood and the administration of drugs. The body temperature was monitored by a rectal thermometer and the animals were maintained at normothermia by external heating.In normotensive animals, if there was a transient reduction in mean arterial blood pressure below 80mm Hg for more than two minutes during the observation period of 3-4 hours after occlusion of the MCA, the animals were not processed for inclusion in the study.Operative procedure The proximal portion of the MCA was occluded permanently under halothane anaesthesi...

Selective neuronal death in the CA1 sector of the hippocampus [delayed neuronal death (DND)] develops several days after transient global cerebral ischemia in rodents. Because NGF plays a potential role in neuronal survival, it was decided to study its effect in DND. We report here that intraventricular injection of NGF either before or after 5 min forebrain ischemia in the Mongolian gerbil significantly reduced the occurrence of DND. The tissue content of NGF in the hippocampus was decreased 2 d after ischemia and recovered to the preischemic level by 1 week. By the Golgi staining technique, changes first began in the dendrites of affected neurons as early as 3 hr. Such changes could be ameliorated by NGF treatment. Although previous knowledge of NGF is limited to the survival of cholinergic neurons in the CNS, it is assumed that other mechanisms must be operating in the hippocampus, for example, postsynaptic modification at dendrites or aberrant expression of NGF receptors possibly at the initial excitation period by glutamate. Furthermore, because previous work has shown that inhibition of protein synthesis reduces the occurrence of DND, a program leading to cell death might also be operating via de novo synthesis of certain protein(s), collectively termed “killer protein,” because of a lack of NGF.

✓ The microgravimetric technique and the drying-weighing method for the determination of brain water content are analyzed and compared. A new method has been devised for the automatic production of the gradient column. For gravimetry, tissue samples weighing more than 30 mg have proven adequate for measurement. Specific gravity (SG) should be determined as early as 1 minute after tissue is inserted into the gradient column. Calculations of cerebral blood volume (CBV) from changes in SG of both brain tissue and intravascular perfusate have shown that the SG of brain tissue is considerably influenced by changes in CBV. This is because the SG of blood is higher than that of brain tissue, and may lead to a decrease of SG of about 0.002 in anemic cortex and of 0.001 in anemic white matter, which will simulate a false increase in tissue volume as water of 4% and 2%, respectively. This methodological error may be relevant when the early stages of ischemic brain edema development are studied. Water content of brain tissue can also be determined with acceptable accuracy by vacuum freeze-drying samples of brain tissue weighing about 100 mg. In contrast to cortex, white matter shows a wide range of individual and regional differences in water content. Thus, conclusions on the presence of brain edema drawn from tissue water determinations should always be subjected to cautious analysis and criticism.

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We investigated the in vivo vasoconstrictor effect of endothelin, a recently characterized vasoconstrictor peptide from vascular endothelium, in the basilar arteries of five cats and five dogs. Basilar artery caliber was angiographically measured under anesthesia before (control) and after either vertebral artery infusion or cisternal injection of the peptide. In cats, 5-500 pmol endothelin induced a dose-dependent basilar artery contraction in vivo when injected intracisternally; within 3 minutes after injection of 500 pmol endothelin, basilar artery caliber was decreased by 73±4% compared with control diameter before injection. The vasoconstriction was extremely long-lasting; no significant recovery of basilar artery caliber was observed for up to 2 hours after injection. In contrast, infusion of up to 3,000 pmol endothelin into the vertebral artery had no appreciable effect on basilar artery caliber. Similar results were obtained in dogs; vasoconstriction was maintained for as long as 12 hours. T he endothelium of cerebral blood vessels is now recognized not only as a physical barrier isolating the brain from the environment but also as an organ having a number of important active functions. 1 -2 Both cerebral and peripheral endothelium are known to produce different classes of diffusible vasoactive factors in response to a variety of stimuli, thereby regulating vascular smooth muscle tonus.3 Endothelin is a vasoconstrictor peptide that we recently characterized from the supernatant of cultured porcine aortic endothelial cells. 4 Consisting of 21 amino acid residues with two intrachain disulfide bonds, endothelin is one of the most potent vasoconstrictors known. Endothelin-induced constriction of cerebral and peripheral arteries from various species in vitro is extremely long-lasting and is characteristically dif- Received May 17, 1989; accepted July 5, 1989. ficult to wash out. In vivo, an intravenous bolus injection of endothelin to anesthetized, conscious rats causes a sustained increase in arterial blood pressure that lasts for >1 hour. Cloning and sequence analysis of endothelin complementary deoxyribonucleic acid from porcine and human endothelial cells has demonstrated that endothelin is produced from an approximately 200-residue prepro-type precursor much like other peptide hormones and that human and mature porcine endothelin are identical. 5 The level of preproendothelin messenger ribonucleic acid (mRNA) in endothelial cells is influenced by various chemical and mechanical stimuli, suggesting the importance of endothelin in the local control of vascular tonus.We are interested in the possible involvement of endothelin in the local regulation of cerebral arteries in vivo. It is of special interest to assume that production of excess endothelin contributes to the development of delayed vasospasm after subarachnoid hemorrhage. We investigated the response of feline and canine basilar artery in vivo to the intra-arterial and intracisternal administration of endothelin.

The development of cerebral edema after experimental subarachnoid hemorrhage (SAH) was studied in cats by determining regional brain tissue water content with the microgravimetric technique as well as the drying-weighing method. SAH was induced by withdrawing needles previously pierced into one or both infraclinoid internal carotid arteries through a unilateral transorbital approach. Serial determinations of regional cerebral blood flow (rCBF) by labelled microspheres, and monitorings of vital signs such as intracranial pressure (ICP), blood pressure and EEG were carried out up to 24 h after SAH. Animals could be classified into three grades according to the severity of SAH. In grade I, the increase of ICP was transient and minor. In grade II, ICP increased up to 200 mm Hg with a marked reduction of rCBF below 20% of control in cerebral hemispheres. Following subsequent reduction of ICP, rCBF increased over control, indicating reactive hyperemia. Thereafter, a great reduction of rCBF was again observed. In grade III, rCBF was sustained at essentially zero flow with the presence of continuously increased ICP above 100 mm Hg. Cerebral edema was observed particularly in the parasagittal water-shed areas of all grade II animals. It is concluded that cerebral edema complicating SAH is caused by the combination of an initially induced global cerebral ischemia and the subsequent recovery of cerebral circulation. Post SAH hypertension is another factor to exacerbate the development of cerebral edema.

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