Maria Spatz

Experimental cerebral ischemia in Mongolian gerbils

SummaryBehaviour of biogenic amines was studied in the brains of Mongolian gerbils subjected to unilateral occlusion of the common carotid artery. Assays on the hemispheres ipsilateral to occlusion revealed in symptom-positive animals a progressive decrease in norepinephrine and dopamine, and an increase in serotonin throughout the duration of an ischemic insult. In post-ischemic periods following the release of the clip, changes in biogenic amine levels generally conformed to the principles of a previously described “maturation” phenomenon, with delayed reactions occurring after the shorter ischemic insults.

Isolation of brain capillaries: a simplified technique.

Few different procedures have been described for the isolation of brain capillaries recently. The reported methods were simple but cumbersome, requiring the repeated use of nylon cloth sieve or glass beads column for the elimination of nonvascular cerebral tissue or debris in order to obtain a pure or relatively pure fraction of capillaries1, 9x°,1~. In this communication we will outline an easy system (based on the techniques of Goldstein et al. and Joo and Karnushina, see refs. 9 and 10) for the separation of a pure cerebral microvascular fraction which is morphologically intact and enzymatically active, as well as suitable for transport studies. Rabbit cerebral tissue (forebrain freed from pia arachnoid membrane) was homogenized in 20 volumes of cold Ringers solution containing 1 ~ bovine serum albumin (SBA, Sigma Chemical Co., St. Louis, Mo.) and 10 m M Hepes (Calbiochem, San Diego, Calif.) at pH 7.4 in a glass homogenizer using a slow speed motor driven pestle. Six to seven upwards and downwards strokes were applied during the homogenization. The homogenate was centrifuged at 1500 x g for 15 min, the supernatant was discarded and the resuspended second pellet in 1 ~ SBA-Ringers solution was again centrifuged at 1500 x g for 10 rain. The third pellet was suspended in 10 ml of cold 0.25 M sucrose at pH 7.0 and layered over (90 min in advanced prepared) 1.0-1.5 M sucrose gradient (each 12 ml) and centrifuged at 58,000 x g using a Spinco SW-27 rotor in Beckman L2-65B ultracentrifuge for 30 min. This procedure yielded three morphologically distinct fractions. Only the pellet containing microvessels was used for further histological and histochemical investigations, as well as for transport studies. The amount of 1 ~ SBA-Ringer solution used for the final suspension of this fraction varied from 1-2 ml depending on the experimental needs.

Endothelin induction of adhesion molecule expression on human brain microvascular endothelial cells.

The adhesion of circulating leukocytes to vascular endothelium is a prerequisite for their emigration to extravascular tissues. The experiments presented here demonstrate that intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are constitutively expressed on cerebromicrovascular endothelial cell lines derived from human brain and that the expression of these molecules can be up-regulated by endothelins (ET-1, ET-2, and ET-3) in a dose- and time-dependent manner. The data also indicate that ET-1 treatment induced the expression of E-selectin on these cells. These findings implicate vasoactive peptides in the recruitment of blood cells at sites of inflammation.

Endocannabinoids and neuroprotection.

Traumatic brain injury (TBI) releases harmful mediators that lead to secondary damage. On the other hand, neuroprotective mediators are also released, and the balance between these classes of mediators determines the final outcome after injury. Recently, it was shown that the endogenous brain cannabinoids anandamide and 2-Arachidonoyl glycerol (2-AG) are also formed after TBI in rat and mouse respectively, and when administered after TBI, they reduce brain damage. In the case of 2-AG, better results are seen when it is administered together with related fatty acid glycerol esters. Significant reduction of brain edema, better clinical recovery, and reduced infarct volume and hippocampal cell death are noted. This new neuroprotective mechanism may involve inhibition of transmitter release and of inflammatory response. 2-AG is also a potent modulator of vascular tone, and counteracts the endothelin (ET-1)-induced vasoconstriction that aggravates brain damage; it may thus help to restore blood supply to the injured brain. Traumatic brain injury (TBI) causes the accumulation of harmful endogenous constituents in the brain, which may lead to secondary damage; protective mechanisms are also set in motion. Understanding the pathophysiology of secondary brain injury might lead to the development of novel therapeutic treatments for this common condition. The synthesis in the brain of the endogenous cannabinoids 2-arachidonoyl glycerol (2-AG) and anandamide is enhanced in mice and rats, respectively, after TBI and reduces brain damage. These compounds represent a new class of neuroprotective agents that have multiple mechanisms of action that may involve inhibition of transmitter release and of the inflammatory response and might improve the blood supply to the injured brain.

Cytokine-regulated adhesion between encephalitogenic T lymphocytes and cerebrovascular endothelial cells

Adhesive interactions between murine cerebrovascular endothelial cells (EC) which comprise the blood-brain barrier (BBB) and myelin basic protein (MBP)-specific encephalitogenic T lymphocytes were investigated. Adhesion was assessed by measuring the percent attachment of 51Cr-labeled T cells to EC monolayers. The basal level adhesion (20-35%) was significantly up-regulated by treating EC with recombinant murine gamma interferon (IFN-gamma), interleukin-1 alpha (IL-1 alpha) and/or tumor necrosis factor-alpha (TNF alpha). The ability of these cytokines to modulate adhesion was dose- and time-dependent and could be detected as early as 1 h after treatment. The expression of intercellular adhesion molecule-1 (ICAM-1) by EC was examined by immunofluorescence staining and ELISA. Although all unstimulated EC cultures expressed ICAM-1, treatment of EC with the above cytokines dramatically up-regulated the level of ICAM-1 expression in a dose- and time-dependent fashion similar to that observed in the adhesion assays. Treatment of EC with transforming growth factor-beta 1 (TGF beta) down-regulated the level of T cell adhesion on untreated EC in a dose-dependent manner. Pretreatment of EC with TGF beta also partially inhibited the up-regulation of adhesion induced by IFN-gamma, IL-1 alpha and/or TNF alpha. TGF beta had no effect on the up-regulation of ICAM-1 expression induced by IFN-gamma, IL-1 alpha and/or TNF alpha. These results indicate that in addition to ICAM-1, other molecules may be involved in adhesion of encephalitogenic T cells to the EC comprising the cerebral vasculature.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplacental Chemical Induction of Microencephaly in Two Strains of Rats. I

Summary An experimental model for successful chemical induction of microencephaly is described. Methylazoxymethanol, the aglycone of cycasin, when administered to rats at a dose of 20 mg/kg of body weight on day 14 or 15 of pregnancy, uniformly produces microencephaly in all litter mates. This effect occurs independent of the strain of rats used and is highly reproducible. The condition is not associated with microsplanchnia and appears to be limited to the cortical hemispheres of the brain. Preliminary pathologic studies indicate a marked cytotoxic effect of the chemical on the neuroblast. Gliomas have occurred in about 10% of microencephalic rats permitted to live beyond 1 year.

Endothelial cell cultures derived from isolated cerenrbal microvessels

The passage of substances across the blood-brain barrier is regulated by cerebral capillaries which possess certain distinctly different morphological and enzymatic properties compared to the capillaries of other organs. The intercellular diffusion is restricted by the tight junction separating the continuous layer of endothelial cells, while the vesicular transport is limited in the normal endothelium. Many harmful conditions such as anoxia, ischemia, metabolic and/or degenerative disturbances may alter the endothelium and thus the barrier function. Therefore the elucidation of mechanisms involved in various cerebrovascular disorders such as cerebral ischemia could be facilitated by the study of cellular components of the cerebral vessels in a living state. The present study was designed to establish a cultured cell line originating from cerebral microvessels. In this communication we will describe the establishment of a cell line culture showing the characteristic cytochemical features of cerebral endothelial cells, and which originated from dissociated cells of isolated cerebral microvessels. The brains of 2-day-old Osborne-Mendel rats (36-40) were used under sterile conditions for the separation of the microvessels from the non-vascular tissue. The cerebral hemispheres were freed of leptomeninges, minced with scissors and homogenized in buffered (10 mm Hepes) 0.1 ~ albumin-Ringer solution at pH 7.4. The fraction of microvessels was obtained by centrifugation and discontinuous sucrose gradient (1.0-1.5 m), a simple technique described previouslylk Two samples of microvessels (pellets) were pooled and washed twice with 10 ml of modified Simms Balanced Salt Solution (BSS) containing 100 U penicillin, 100/~g streptomycin and 0.25 #g Fungizone (Gibco, Long Island, N.Y.). They were centrifuged immediately after the first wash and then washed again for 30 min in the refrigerator prior to centrifugation at

L-arginine induces dopamine release from the striatum in vivo

Recent reports indicate that induction of nitric oxide (NO) evokes dopamine (DA) release from the striatum in vitro. In this study, we used L-arginine (L-Arg) to demonstrate the in vivo stimulation of DA release from the striatum of Mongolian gerbils using microdialysis. The content of DA in the striatal extracellular fluid (ECF) increased 7–15-fold in the presence of L-Arg in the perfusate as compared with that of the controls (DA level in drug-free perfusate varied from 0.050 ± 0.009 to 0.092 ± 0.023 pmol 10 μUl−1). Simultaneous perfusion of L-Arg with nitro-L-arginine (NLA), an inhibitor of nitric oxide synthase, markedly reduced the L-Arg effect on DA release from the striatum. The NLA-perfused animals contained DA levels significantly lower than those observed in the control striatal dialysate. These findings indicate for the first time that DA release in vivo can be induced by L-Arg, the precursor of NO. The data strongly suggest that NO may modulate striatal DA release.

Intellectual Deficit Associated with Transplacentally Induced Microcephaly in the Rat

Fischer rats injected with methylazoxymethanol late in pregnancy produce young with considerably reduced cerebral hemispheres. They appear normal otherwise. As adults these animals make many more errors in the Hebb-Williams maze than do control animals.

QUANTITATIVE CHANGES WITH AGE IN THE DNA CONTENT OF METHYLAZOXYMETHANOL-INDUCED MICROENCEPHALIC RAT BRAIN

Abstract— DNA synthesis in methylazoxymethanol (MAM)‐treated foetal brain was reduced during the first 3 days after the injection of the compound into the mother rat. The MAM‐treated brain grew at almost the normal rate after this period, but the reduction in DNA persisted through maturity of the animal. This difference in DNA content between normal and microencephalic brain was restricted to the cerebral hemispheres. The major increase in DNA content of prenatal brain occurred in the cerebrum, whereas the postnatal increase took place in the cerebellum. jH‐Labelled MAM was incorporated more extensively into foetal brain DNA than into RNA. The half‐life of the MAM‐modified nucleic acids was 4–5 days. We suggest that removal of necrotic cells from the brain may account for the rapid loss of label from nucleic acids.