Nabil A. Azzam

Mice lacking GFAP are hypersensitive to traumatic cerebrospinal injury

GLIAL fibrillary acidic protein (GFAP) is an intermediate filament protein expressed primarily in astrocytes. We have tested whether GFAP protects against mechanical stress by inducing percussive head injury in GFAP-null mice with a weight drop device. When mice were positioned on a foam bed which allowed head movement at impact, all 14 wild-type mice tested survived, but 12 of 15 GFAP-null mice died within a few minutes. The cause of death appeared to be upper cervical spinal cord injury resulting in respiratory arrest. When the foam bed was replaced by a firm support, both GFAP-null and wild-type mice survived. These results indicate that mice lacking GFAP are hypersensitive to cervical spinal cord injury caused by sudden acceleration of the head.

TNF-α pretreatment prevents subsequent activation of cultured brain cells with TNF-α and hypoxia via ceramide

We have developed a cellular model in which cultured astrocytes and brain capillary endothelial cells preconditioned with tumor necrosis factor-α (TNF-α) fail to upregulate intercellular adhesion molecule-1 (ICAM-1) protein (80% inhibition) and mRNA (30% inhibition) when challenged with TNF-α or exposed to hypoxia. Inasmuch as ceramide is known to mediate some of the effects of TNF-α, its levels were measured at various times after the TNF-α preconditioning. We present evidence for the first time that, in normal brain cells, TNF-α pretreatment causes a biphasic increase of ceramide levels: an early peak at 15-20 min, when ceramide levels increased 1.9-fold in astrocytes and 2.7-fold in rat brain capillary endothelial cells, and a delayed 2- to 3-fold ceramide increase that occurs 18-24 h after addition of TNF-α. The following findings indicate that the delayed ceramide accumulation results in cell unresponsiveness to TNF-α: 1) coincident timing of the ceramide peak and the tolerance period, 2) mimicking of preconditioning by addition of exogenous ceramide, and 3) attenuation of preconditioning by fumonisin B1, an inhibitor of ceramide synthesis. In contrast to observations in transformed cell lines, the delayed ceramide increase was transient and did not induce apoptosis in brain cells.

Human Brain Capillary Endothelium: 2-Arachidonoglycerol (Endocannabinoid) Interacts With Endothelin-1

In brain, the regulatory mechanism of the endothelial reactivity to nitric oxide and endothelin-1 may involve Ca2+, cytoskeleton, and vasodilator-stimulated phosphoprotein changes mediated by the cGMP/cGMP kinase system. 1 Endothelium of human brain capillaries or microvessels is used to examine the interplay of endothelin-1 with the putative vasorelaxant 2-arachidonoyl glycerol, an endogenous cannabimimetic derivative of arachidonic acid. This study demonstrates that 2-arachidonoyl glycerol counteracts Ca2+ mobilization and cytoskeleton rearrangement induced by endothelin-1. This event is independent of nitric oxide, cyclooxygenase, and lipoxygenase and is mediated in part by cannabimimetic CB1 receptor, G protein, phosphoinositol signal transduction pathway, and Ca2+-activated K+ channels. The induced rearrangements of cellular cytoskeleton (actin or vimentin) are partly prevented by inhibition of protein kinase C or high levels of potassium chloride. The 2-arachidonoyl glycerol–induced phosphorylation of vasodilator-stimulated phosphoprotein is mediated by cAMP. These findings suggest that 2-arachidonoyl glycerol may contribute to the regulation of cerebral capillary and microvascular function.

Nitric Oxide Modulates Endothelin 1-Induced Ca2+ Mobilization and Cytoskeletal F-Actin Filaments in Human Cerebromicrovascular Endothelial Cells

A functional interrelation between nitric oxide (NO), the endothelial-derived vasodilating factor, and endothelin 1 (ET-1), the potent vasoconstrictive peptide, was investigated in microvascular endothelium of human brain. Nor-1 dose-dependently decreased the ET-1–stimulated mobilization of Ca2+. This response was mimicked with cGMP and abrogated by inhibitors of guanylyl cyclase or cGMP-dependent protein kinase G. These findings indicate that NO and ET-1 interactions involved in modulation of intracellular Ca2+ are mediated by cGMP/protein kinase G. In addition, Nor-1–mediated effects were associated with rearrangements of cytoskeleton F-actin filaments. The results suggest mechanisms by which NO–ET-1 interactions may contribute to regulation of microvascular function.

Regenerating Axons Are Not Required to Induce the Formation of a Schwann Cell Cable in a Silicone Chamber

After suture of proximal and distal nerve stumps into the ends of a silicone chamber, a tissue cable forms inside the chamber through which axons regenerate. Schwann cells are a critical cellular component of the cable because in their absence axons fail to regenerate into the cable. In this study, we sought to determine whether axons were needed to induce the formation of a Schwann cell-containing cable. Transected stumps of sciatic nerves of adult rats were sutured into the ends of silicone chambers prefilled with phosphate-buffered saline or dialyzed plasma, leaving a 10-mm interstump gap. In order to eliminate any axonal influence in the chamber, the proximal sciatic nerve was further transected, ligated, and reflected, leaving a 4-mm piece of denervated nerve in the proximal chamber. A tissue cable formed at 4 weeks only in those chambers prefilled with dialyzed plasma. Light and electron microscopy revealed a central core of Schwann cells and fibroblasts within the cable that were collectively surrounded by a circumferential layer of fibroblasts and collagen. Blood vessels were randomly located throughout the cable. The Schwann cells extended numerous processes that were confined within a basal lamina-like membrane. Many of these processes contained microtubules and resembled unmyelinated axons. The ultrastructure of the processes, however, differed from that of axons in that some of the processes were in direct contact with the basal lamina of the Schwann cells and not surrounded by any other cell extensions. However, since these processes neither stained with silver nor disappeared after transection of the nerves entering or leaving the chamber, we conclude that they are not axons but in fact Schwann cell processes. In other animals bearing 4-week cables, the reflected nerve stump was reattached to the nerve piece in the proximal end of the chamber. Four weeks later, all the cables and varying lengths of the distal nerve trunks were filled with numerous myelinated and unmyelinated axons. The Schwann cell cable that forms within a dialyzed plasma prefilled chamber presents a useful system for basic research concerning the molecular mechanisms of Schwann cell or Schwann cell-axonal interactions and for applied research involving the clinical repair of human peripheral nerve injuries. Since a cable formed by our surgical method supports axonal regeneration, it has the potential to eliminate the need for a nerve graft to repair a gap in a nerve that requires delayed surgical intervention.

Membrane changes during hibernation.

Cellular membranes are susceptible to injury by cold, which causes their lipid components to separate. Here we investigate the structural changes that occur in organelle membranes inside cells of the central nervous system of hypothermic ground squirrels during hibernation. We find that lipids in these membranes sequester into protein-free domains that laterally displace membrane proteins and the underlying cytoplasmic matrix. But when the animal is aroused from hibernation, all these components return to their normal arrangements as the body temperature rises. Understanding this reversible temperature-induced redistribution of membrane lipids should help in the study of the effects of severe cold on non-hibernating species (including humans) and in the cryopreservation of cells and tissues.

Endothelin-1 and Nitric Oxide Affect Human Cerebromicrovascular Endothelial Responses and Signal Transduction

Endothelium plays a central role in regulating the vascular tone, blood flow and blood brain barrier (BBB) permeability. The experiments presented here examine the mechanisms by which nitric oxide (NO) and endothelin-1 (ET-1) may be involved in these processes. The findings indicate that ET-1-stimulated [Ca2+]i accumulation occurs through activation of ETA receptor. The capacity of NO to affect this response was indicated by results showing: 1) a two-fold increase in ET-1-stimulated [Ca2+]i by L-NAME, the inhibitor of nitric oxide synthase, and 2) a dose-dependent decrease in [Ca2+]i accumulation by pretreatment with Nor-1 (NO donor). Abrogation of this Nor-1 effect by ODQ (an inhibitor of guanylyl cyclase) or Rp-8-pCPT-cGMPS (an inhibitor of protein kinase G) and inhibition of ET-1 stimulated intracellular Ca2+ accumulation by 8-bromo-cGMP (a permeable, analog of cGMP) substantiate the involvement of interplay between ET-1 and NO in [Ca2+]i accumulation in HBMEC. ET-1 treatment also increased thickness of F-actin cytoskeletal filaments in HBMEC. This effect was attenuated by pretreatment with NO; NO also rarefied F-actin filaments in control cultures. The findings support a linkage between NO and ET-1 in regulating microvascular tone, microcirculation and BBB permeability and indicate a role for cGMP/cGMP protein kinase system and cytoskeletal changes in responses of HBMEC.

The Loss of Regenerated Host Axons in Nerve Allografts after Stopping Immunosuppression with Cyclosporin A Is Related to Immune Effects on Allogeneic Schwann Cells

After immunosuppressive therapy with Cyclosporin A (Cy-A) is stopped, nerve allograft rejection occurs. In addition to the loss of allogeneic perineurial, vascular, and Schwann cells, host axons that regenerate into the allograft disappear despite the fact that the axons are not foreign tissue. The present experiment was performed to correlate immune events and allogeneic cell and host axonal loss in nerve allografts after terminating Cy-A treatment. Nerve grafts (4 cm long) were taken from American Cancer Institute (ACI) rats and joined to the peroneal nerves of Fischer (FR) or ACI rats that received a daily dose of Cy-A (10 mg/kg, intraperitoneally). After one week, Cy-A therapy was stopped and the grafts were examined 2-6 weeks postoperatively by light and electron microscopy. No immune reaction nor destruction of perineural, vascular, or Schwann cells was found in 2- or 3-week-old allografts (i.e., ACI to FR grafts). These grafts underwent Wallerian degeneration and were invaded proximally by regenerating host axons, some of which were thinly myelinated. At 4 weeks, the perineurium of each allograft became infiltrated by mononuclear cells and was destroyed. Many of the endoneurial blood vessels of these grafts were occluded and their endothelial cells were degenerating or missing. Despite the immune reaction, allogeneic Schwann cells remained and continued to myelinate or ensheath host axons that had now grown up to 3 cm into the grafts.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of Tyrosine Phosphorylation of a Brain Protein by Hibernation

Mammalian hibernation is a state of natural tolerance to severely decreased brain blood flow. As protein tyrosine phosphorylation is believed to be involved in the development of resistance to potentially cell-damaging insults, we used immunoblotting for the phosphotyrosine moiety to analyze extracts from various tissues of hibernating and nonhibernating ground squirrels. A single, hibernation-specific phosphoprotein was detected in the brain, but not in any other tissue tested. This protein, designated pp98 to reflect its apparent molecular weight, is distributed throughout the brain, and is associated with the cellular membrane fraction. The presence of the protein is tightly linked to the hibernation state; it is not present in contemporaneously assayed animals that are exposed to the same cold temperature as the hibernators, is present for the duration of a hibernation bout (tested from 1 to 14 days), and disappears within 1 hour of arousal from hibernation. The close association of pp98 with the hibernation state, its presence in cellular membranes, and the known properties of membrane phosphotyrosine proteins suggest that it may transduce a signal for adaptation to the limited availability of oxygen and glucose and low cellular temperature that characterizes hibernation in the ground squirrel.

Host nerve fibers that regenerate and reside long-term in a rejected nerve allograft are not protected by permeability barriers

We investigated whether permeability barriers develop and protect host nerve fibers that regenerate and reside long-term in a rejected nerve allograft. In order for barriers to form, host cells have to enter the rejected allograft and differentiate into new endothelial and perineurial cells that respectively form the impermeable endoneurial blood-nerve and the perineurium-nerve barriers that are present in normal nerve. A 2-cm long graft of peroneal nerve was taken from American Cancer Institute (ACI) or Fischer (FR) rats and transplanted to bridge a 2-cm gap between the cut ends of the peroneal nerve of other FR rats. Six months postoperatively, histology revealed that regenerated host nerve fibers in ACI allografts were compartmentalized into numerous minifascicles by perineurial cells and that blood vessels were located outside rather than inside the perineurial compartments among the nerve fibers. Administration of the permeability indicator horseradish peroxidase to allograft recipients (intravenously or topically to the graft in situ) revealed that it entered the endoneurium of microcompartments and spread around the nerve fibers. In contrast, none of the indicator reached nerve fibers in FR syngrafts or normal ACI or FR nerves which were not microcompartmentalized. We concluded that host nerve fibers that regenerate and reside long-term in a rejected nerve allograft are not protected by permeability barriers.