Ronald F. Tuma

Clinical Trials in Head Injury

Secondary brain damage, following severe head injury is considered to be a major cause for bad outcome. Impressive reductions of the extent of brain damage in experimental studies have raised high expectations for cerebral neuroprotective treatment, in the clinic. Therefore multiple compounds were and are being evaluated in trials. In this review we discuss the pathomechanisms of traumatic brain damage, based upon their clinical importance. The role of hypothermia, mannitol, barbiturates, steroids, free radical scavengers, arachidonic acid inhibitors, calcium channel blockers, N-methyl-D-aspartate (NMDA) antagonists, and potassium channel blockers, will be discussed. The importance of a uniform strategic approach for evaluation of potentially interesting new compounds in clinical trials, to ameliorate outcome in patients with severe head injury, is proposed. To achieve this goal, two nonprofit organizations were founded: the European Brain Injury Consortium (EBIC) and the American Brain Injury Consortium (ABIC). Their aim lies in conducting better clinical trials, which incorporate lessons learned from previous trials, such that the succession of negative, or incomplete studies, as performed in previous years, will cease.

The Proinflammatory Effect of Prostaglandin E2 in Experimental Inflammatory Bowel Disease Is Mediated through the IL-23→IL-17 Axis

Although Crohn’s disease has been traditionally considered to be Th1-mediated, the newly identified Th17 cells emerged recently as crucial participants. Th1/Th17 differentiation is controlled primarily by the IL-12 family of cytokines secreted by activated dendritic cells (DCs) and macrophages. IL-23 and IL-12/IL-27 have opposite effects, supporting the Th17 and Th1 phenotypes, respectively. We found that PGE2, a major lipid mediator released in inflammatory conditions, shifts the IL-12/IL-23 balance in DCs in favor of IL-23, and propose that high levels of PGE2 exacerbate the inflammatory process in inflammatory bowel disease through the IL-23→IL-17 axis. We assessed the effects of PGE2 on IL-12, IL-27, and IL-23 and found that PGE2 promotes IL-23, inhibits IL-12 and IL-27 expression and release from stimulated DCs, and subsequently induces IL-17 production in activated T cells. The effects of PGE2 are mediated through the EP2/EP4 receptors on DCs. In vivo, we assessed the effects of PGE analogs in an experimental model for inflammatory bowel disease and found that the exacerbation of clinical symptoms and histopathology correlated with an increase in IL-23 and IL-17, a decrease in IL-12p35 expression in colon and mesenteric lymph nodes, and a substantial increase in the number of infiltrating neutrophils and of CD4+IL-17+ T cells in the colonic tissue. These studies suggest that high levels of PGE2 exacerbate the inflammatory process through the preferential expression and release of DC-derived IL-23 and the subsequent support of the autoreactive/inflammatory Th17 phenotype.

Influence of oxygen on perfused capillary density and capillary red cell velocity in rabbit skeletal muscle

Abstract The pattern of capillary blood flow changes was investigated in rabbit tenuissimus muscle as a function of oxygen tension in the solution suffusing the muscle. The density of perfused capillaries decreased from 269 ± 22 (mean ± SD) to 6 ± 10 cap/mm 2 when ambient p O 2 was elevated progressively from 5 to 100 mm Hg. In the same p O 2 range capillary red cell velocity decreased from 0.29 ± 0.14 (mean ± SD) to 0.18 ± 0.06 mm/sec in the capillaries which were perfused. During exposure of the muscle to atmospheric oxygen tension ( p O 2 = 150 mm Hg) there were no capillaries perfused and hence flow velocity was zero in all capillaries. The results demonstrate that oxygen influences capillary blood flow over an extensive range of oxygen tensions. The anatomical structures responsible for the alterations in both capillary density and capillary flow velocity are the arterioles. This site of control determines actively the total blood flow through the capillary bed, whereas passive factors seem to determine the distribution of the flow between specific capillaries. The results do not support the existence of precapillary sphincters controlling perfused capillary density.

Cannabinoid CB2 receptor activation decreases cerebral infarction in a mouse focal ischemia/ reperfusion model

Cannabinoid CB2 Receptor (CB2) activation has been shown to have immunomodulatory properties without psychotropic effects. The hypothesis of this study is that selective CB2 agonist treatment can attenuate cerebral ischemia/reperfusion injury. Selective CB2 agonists (O-3853, O-1966) were administered intravenously 1 h before transient middle cerebral artery occlusion (MCAO) or 10 mins after reperfusion in male mice. Leukocyte/endothelial interactions were evaluated before MCAO, 1 h after MCAO, and 24 h after MCAO via a closed cranial window. Cerebral infarct volume and motor function were determined 24 h after MCAO. Administration of the selective CB2 agonists significantly decreased cerebral infarction (30%) and improved motor function (P < 0.05) after 1 h MCAO followed by 23 h reperfusion in mice. Transient ischemia in untreated animals was associated with a significant increase in leukocyte rolling and adhesion on both venules and arterioles (P < 0.05), whereas the enhanced rolling and adhesion were attenuated by both selective CB2 agonists administered either at 1 h before or after MCAO (P < 0.05). CB2 activation is associated with a reduction in white blood cell rolling and adhesion along cerebral vascular endothelial cells, a reduction in infarct size, and improved motor function after transient focal ischemia.

Activation of Cannabinoid Receptor 2 Attenuates Leukocyte–Endothelial Cell Interactions and Blood–Brain Barrier Dysfunction under Inflammatory Conditions

Previous studies have shown that modulation of the receptor-mediated cannabinoid system during neuroinflammation can produce potent neuroprotective and anti-inflammatory effects. However, in this context, little is known about how selective activation of the cannabinoid type-2 receptor (CB2R) affects the activated state of the brain endothelium and blood–brain barrier (BBB) function. Using human brain tissues and primary human brain microvascular endothelial cells (BMVECs), we demonstrate that the CB2R is highly upregulated during inflammatory insult. We then examined whether the CB2R agonists could attenuate inflammatory responses at the BBB using a mouse model of LPS-induced encephalitis and highly selective CB2R agonists. Visualization by intravital microscopy revealed that administration of JWH133 [(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran] or a novel resorcinol-based compound, O-1966 (1-[4-(1,1-dimethyl-heptyl)-2,6-dimethoxy-phenyl]-3-methyl-cyclohexanol), greatly attenuated leukocyte adhesion in surface pial vessels and in deep ascending cortical postcapillary venules. BBB permeability assessments with small and large fluorescent tracers showed that CB2R agonists were effective at preventing barrier leakiness after LPS administration. To determine whether the effects by CB2R agonists on barrier protection are not only due to the CB2R modulation of immune cell function, we tested the agonists in vitro with barrier-forming primary BMVECs. Remarkably, the addition of CB2R agonist increased transendothelial electrical resistance and increased the amount of tight junction protein present in membrane fractions. Furthermore, CB2R agonists decreased the induction of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 surface expression in BMVECs exposed to various proinflammatory mediators. Together, these results suggest that pharmacological CB2R ligands offer a new strategy for BBB protection during neuroinflammation.

Win 55212-2, a cannabinoid receptor agonist, attenuates leukocyte/endothelial interactions in an experimental autoimmune encephalomyelitis model

Multiple sclerosis (MS) is the most common of the immune demyelinating disorders of the central nervous system (C NS). Leukocyte/endothelial interactions are important steps in the progression of the disease and substances that interfere with these activities have been evaluated as potential therapeutic agents. C annabinoid receptor agonists have been shown to downregulate immune responses and there is preliminary evidence that they may slow the progress of MS. The purpo se of this investigation was to determine how cannabinoid recepto r agonists interfere with leukocyte rolling and adhesion. This was investigated in an experimental autoimmune encephalo myelitis (EAE) model using six to eight week old C 57BL/6 mice. Mouse myelin oligodendrocyte protein and pertussis toxin were used to induce EAE. WIN 55212-2, C B1 and C B2 antagonist were given. By use of in vivo intravital microscopy, leukocyte/endothelial interactio ns were evaluated via a cranial window implanted two days before. The results demonstrated that EAE increases leukocyte rolling and firm adhesion in the brain, and that this increased leukocyte/endothelial interactio n can be attenuated by administration of WIN 55212-2. Furthermore, use of the selective antagonists for the C B1 recepto r (SR 141716A) and the C B2 receptor (SR144528) in this study demonstrated that the cannabinoid’s inhibitory effects on leukocyte/endothelial interactions can be mediated by activating C B2 receptor.

SB 239063, a novel p38 inhibitor, attenuates early neuronal injury following ischemia.

The aim of the present study was to evaluate p38 MAPK activation following focal stroke and determine whether SB 239063, a novel second generation p38 inhibitor, would directly attenuate early neuronal injury. Following permanent middle cerebral artery occlusion (MCAO), brains were dissected into ischemic and non-ischemic cortices and Western blots were employed to measure p38 MAPK activation. Neurologic deficit and MR imaging were utilized at various time points following MCAO to monitor the development and resolution of brain injury. Following MCAO, there was an early (15 min) activation of p38 MAPK (2.3-fold) which remained elevated up to 1 h (1.8-fold) post injury compared to non-ischemic and sham operated tissue. Oral SB 239063 (5, 15, 30, 60 mg/kg) administered to each animal 1 h pre- and 6 h post MCAO provided significant (P<0.05) dose-related neuroprotection reducing infarct size by 42, 48, 29 and 14%, respectively. The most effective dose (15 mg/kg) was further evaluated in detail and SB 239063 significantly (P<0.05) reduced neurologic deficit and infarct size by at least 30% from 24 h through at least 1 week. Early (i.e. observed within 2 h) reductions in diffusion weighted imaging (DWI) intensity following treatment with SB 239063 correlated (r=0.74, P<0.01) to neuroprotection seen up to 7 days post stroke. Since increased protein levels for various pro-inflammatory cytokines cannot be detected prior to 2 h in this stroke model, the early improvements due to p38 inhibition, observed using DWI, demonstrate that p38 inhibition can be neuroprotective through direct effects on ischemic brain cells, in addition to effects on inflammation.

Leukocyte involvement in cerebral infarct generation after ischemia and reperfusion

White blood cell involvement in the generation of cerebral infarcts was evaluated following ischemia and reperfusion injury in the rat. Control and leukopenic rats (induced by vinblastine, WBC counts < 1500/mm3) were compared in a global forebrain ischemic model after 1 h of ischemia and 1 h 15 min of reperfusion. Cerebral infarcts were defined on coronal brain sections using Triphenyl tetrazolium chloride (TTC) staining. Electroencephalographic activity (EEG) and somatosensory evoked potentials (SSEP) were also compared. Results indicate that the area infarcted in leukopenic rats was significantly less than infarcts generated in corresponding controls (21 +/- 16% vs. 70 +/- 16%). In addition, EEG was preserved in all leukopenic animals when compared to controls, both during ischemia and after reperfusion. The cortical peak component of the SSEP was also better preserved in the leukopenic animals both during ischemia and at 60 min of reperfusion. These results indicate white blood cell participation in the generation of cerebral damage in a model of global forebrain ischemia and reperfusion as indicated by TTC staining of cerebral infarcts.

Leukocyte involvement in cerebral ischemia and reperfusion injury

Leukocytes have been postulated to contribute to cerebral ischemia and reperfusion injury. The present study implies that leukocytes have a deleterious effect in the brain following ischemia. We compared the alteration of cortical electrical activity following transient, incomplete cerebral ischemia in control and leukopenic rats by monitoring somatosensory evoked potentials and electroencephalographic activity. There was complete cessation of electroencephalographic activity, and the cortical peak of the evoked potential was abolished during ischemia in the control animals. However, when the animals were rendered leukopenic, there was maintenance of electroencephalographic activity with reduced amplitude and preservation of the cortical peak of the evoked response during the ischemic period. This indicates that when the animals are made leukopenic, even under ischemic conditions, the neurophysiologic functioning is still maintained to a certain extent.

Quantitative changes in interleukin proteins following focal stroke in the rat.

The aim of the present study was to quantitate the temporal changes in protein concentration for interleukin (IL)-1alpha, IL-1beta, IL-1ra, and IL-6 from 1 h to 15 days following focal ischemia. Protein expression was evaluated by enzyme-linked immunosorbent assay utilizing newly available rat antibodies. There were no detectable basal levels of IL-1alpha, 1L-1beta, or IL-6 in the sham-operated or non-ischemic control cortex. IL-1beta (increased significantly (P<0.05) as early as 4 h and peaked at 3 to 5 days. IL-1alpha (increased significantly (P<0.05) at 3 days. IL-6 increased early and peaked at 24 h (P<0.05). IL-1ra increased significantly (P<0.05) over basal levels from 12 h to 5 days. The present study provides the first quantitative determination of interleukin protein concentrations in the rat brain following focal stroke and demonstrates that this technology is now available for mechanistic studies in neuroprotection.