John B. Schweitzer

DNA end labeling (TUNEL) in Huntington's disease and other neuropathological conditions.

Deoxyribonucleic acid of cells undergoing apoptosis is cleaved by a calcium-dependent endonuclease into oligonucleosomal-sized fragments. These fragments can be labeled using the enzyme terminal deoxynucleotidyl transferase so that the cells can be visualized immunohistochemically. Few investigators have evaluated this method in disease processes of the human central nervous system. The Tdt-mediated dUTP-biotin nick end labeling (TUNEL) technique has been investigated in preliminary studies of a variety of pathologic conditions of the human brain (e.g., gliomas, traumatic brain injury, Parkinsons disease, Parkinsons-Alzheimers complex, multisystem atrophy, striatonigral degeneration). We focus, however, on Huntingtons disease (HD) because of the availability of well-characterized pathological stages for study, and also because of the neurodegenerative diseases studied to date, only Huntingtons disease revealed significant and consistent labeling with this method. This implies a possibly unique nature to the mechanism of cell death in Huntingtons disease compared to the other neurodegenerative diseases studied. TUNEL+ neurons were found in Grade 1-4 HD neostriatum, while labeled astrocytes were found predominantly in the Grade 1 and 2 cases studied to date. TUNEL+ cells were also found in glioblastoma multiforme and traumatic brain injury. We conclude that while there appear to be several limitations associated with this technique, it may be useful for identifying both apoptosis and necrosis in certain neuropathological conditions.

A grading system for mesial temporal pathology (hippocampal sclerosis) from anterior temporal lobectomy

A practical grading system based on routine light microscopic examination that quantifies histopathlogy of mesial temporal damage in temporal obectomy specimens is proposed. The aim of this grading system is to standardize the terminology used in reporting mesial temporal pathology that will help provide a means of comparing patient populations in research reports. This proposed system has five tiers that range from no hippocampal pathology present to Grade IV (marked hippocampal sclerosis) with three intermediate grades. Our internal blinded testing shows this grading system to be reliable and reproducible when used independently among individual pathologists.

Modulation of the Phosphoinositide 3-Kinase Pathway Alters Innate Resistance to Polymicrobial Sepsis

We examined the effect of modulating phosphoinositide 3-kinase (PI3K) activity in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. Inhibition of PI3K activity with wortmannin increased serum cytokine levels and decreased survival time in septic mice. We have reported that an immunomodulator, glucan phosphate, induces protection in murine polymicrobial sepsis. We observed that glucan stimulated tissue PI3K activity, which positively correlated with increased survival in septic mice. We investigated the effect of PI3K inhibition on survival in septic mice treated with glucan. Treatment of mice with the PI3K inhibitors, wortmannin and LY294002, completely eliminated the protective effect of glucan, indicating that protection against septic mortality was mediated through PI3K. Inhibition of PI3K resulted in increased serum levels of IL1-β, IL-2, IL-6, IL-10, IL-12, and TNF-α in septic mice. Apoptosis is thought to play a central role in the response to septic injury. We observed that inhibition of PI3K activity in septic mice resulted in increased splenocyte apoptosis and a change in the anatomic distribution of splenocyte apoptosis. We conclude that PI3K is a compensatory mechanism that suppresses proinflammatory and apoptotic processes in response to sepsis and/or inflammatory injury. Thus, PI3K may play a pivotal role in the maintenance of homeostasis and the integrity of the immune response during sepsis. We also observed that glucan phosphate decreased septic morbidity and mortality through a PI3K-dependent mechanism. This suggests that stimulation of the PI3K pathway may be an effective approach for preventing or treating sepsis and/or septic shock.

Specificity of 192 IgG-saporin for NGF receptor-positive cholinergic basal forebrain neurons in the rat ☆

A monoclonal antibody to the rat nerve growth factor (NGF) receptor, 192 IgG, accumulates bilaterally and specifically in cholinergic basal forebrain (CBF) cells following intraventricular injection. An immunotoxin composed of 192 IgG linked to saporin (192 IgG-saporin) has been shown to destroy cholinergic neurons in the basal forebrain. We sought to determine if intraventricular 192 IgG-saporin affected choline acetyltransferase (ChAT) enzyme activity in the CBF terminal projection fields. ChAT assays from 192 IgG-saporin-treated animals showed significant time-dependent decreases in ChAT activity in the neocortex, olfactory bulb and hippocampus, compared to PBS- or OKT1-saporin-injected controls. ChAT and tyrosine hydroxylase activity in the striatum was always unchanged by 192 IgG-saporin. ChAT immunohistochemistry was confirmative of major cell loss in the CBF, while other cholinergic nuclei appeared unremarkable. The data provide further evidence of the selectivity of 192 IgG-saporin in abolishing cholinergic, NGF receptor-positive CNS neurons.

Modulation of tissue Toll-like receptor 2 and 4 during the early phases of polymicrobial sepsis correlates with mortality.

OBJECTIVE To determine whether there was a correlation between induction of polymicrobial sepsis, modulation of tissue Toll-like receptor (TLR) gene, and protein expression and survival outcome. DESIGN Prospective, randomized animal study. SETTING University medical school research laboratory. SUBJECTS Age- and weight-matched ICR/HSD mice. INTERVENTIONS Sepsis was induced by cecal ligation and puncture (CLP). No-surgery and sham (laparotomy)-operated mice were controls. We also examined tissue TLR2 and TLR4 messenger RNA and TLR4 protein levels in mice treated with an immunomodulator that increases survival in polymicrobial sepsis. In the immunomodulator study, mice were treated with glucan phosphate (50 mg/kg, intraperitoneally) 1 hr before CLP. No-surgery, sham surgery, glucan + no-surgery, sham surgery + glucan, and CLP groups were employed as controls. MEASUREMENTS AND MAIN RESULTS Total RNA was isolated from liver, lung, and spleen at 0, 1, 3, 6, 8, and 24 hrs after CLP. TLR gene expression was assessed by reverse transcription-polymerase chain reaction. Tissue TLR4 protein levels were evaluated at 24 hrs by Western blot and immunohistochemistry. CLP sepsis increased (p <.05) liver and lung TLR2 and TLR4 gene expression compared with controls. TLR4 protein concentrations also were increased. Increased TLR2/4 gene and TLR4 protein expression correlated with mortality. Immunoprophylaxis with glucan phosphate increased (p <.001) long-term survival (20% vs. 70%) but inhibited (p <.05) CLP-induced increases in tissue TLR2 and TLR4 messenger RNA expression as well as TLR4 protein expression. CONCLUSIONS Early increases in TLR2/4 gene and TLR4 protein expression correlated with mortality, whereas blunting TLR gene and protein expression correlated with improved long-term survival. This suggests that early up-regulation of tissue TLR2/4 may play a role in the proinflammatory response and pathophysiology of polymicrobial sepsis.

Effects of cholinergic depletion on experience-dependent plasticity in the cortex of the rat.

Clinical and functional studies have strongly suggested that acetylcholine input from the nucleus basalis of Meynert is important for the cortexs adaptive response to experience. The purpose of this study was to investigate the effects of depletion of acetylcholine inputs from nucleus basalis of Meynert on experience-dependent plasticity in the cortex of young adult male rats. The posteromedial barrel subfield in the primary somatosensory cortex was studied. Experience-dependent plasticity was elicited using a whisker-pairing paradigm in which all whiskers except D2 and D3 were trimmed daily. Plasticity within barrel D2 of the posteromedial barrel subfield was measured using the electrophysiological extracellular recording technique. An index of plasticity was determined in two ways: as an increase in the magnitude of evoked activity to stimulation of whisker D2 and as a bias in the ratio of evoked activity for stimulation of paired whisker D3 and cut whisker D1 (D3/D1). Whiskers D2, D3 and D1 were stimulated (deflected) by a Chubbuck electromechanical stimulator. Cholinergic neurons in the nucleus basalis of Meynert were selectively lesioned with an immunotoxin, 192 IgG-saporin, injected into the left lateral ventricle. Lesions of cholinergic neurons in the nucleus basalis of Meynert were verified using choline acetyltransferase immunocytochemistry and radioenzymatic assay. Experience-dependent plasticity was significantly reduced in cholinergic-depleted animals. The magnitude of evoked activity to stimulation of whisker D2 increased by 16-100% in control animals compared with 0-20% in cholinergic-depleted animals. Similarly, compared to a 60-100% increase in the D3/D1 ratio of evoked activity for phosphate-buffered saline-injected control animals, cholinergic-depleted rats showed no significant increase in the D3/D1 ratio (0-15%) after undergoing the whisker-pairing paradigm. After whisker trimming, the D3/D1 response ratio in immunotoxin-treated animals was essentially the same as in control animals that had not been subjected to the whisker-pairing paradigm. This study showed that no significant plasticity response was observed in the absence of cholinergic input from the nucleus basalis of Meynert. The mechanisms of the action of acetylcholine in cortical plasticity are still not known, but we hypothesize that this type of plasticity is activity dependent and is significantly enhanced in the presence of acetylcholine.

192 IgG-saporin. I: Specific lethality for cholinergic neurons in the basal forebrain of the rat

An immunotoxin (IT) composed of a monoclonal antibody to the nerve growth factor (NGF) receptor, 192 IgG, chemically linked to saporin, 192 IgG-saporin, was shown to selectively reduce forebrain choline acetyltransferase (ChAT) activity in the rat brain following intraventricular administration. In order to determine if the IT was killing NGF receptorpositive neurons in the CBF (rather than simply suppressing the cholinergic phenotype in these cells), a population of neurons in the nucleus basalis magnocellularis (NBM) was prelabeled by an intracortical injection of the neurotracer Fluoro-Gold (FG) 1 week before intraventricular injections of IT or control substances (reduced IT or phosphate-buffered saline). We found that there were very few double-labeled (i.e. FG-labeled and ChAT-positive) neurons remaining in the NBM of ITtreated animals. The absolute number of FG-labeled neurons in the NBM of IT-treated animals was reduced by a number similar to the counts of double-labeled neurons in the NBM of control animals. Our conclusion is that the IT is preferentially lethal to cholinergic neurons in the NBM. Due to its ability to selectively kill cholinergic neurons in the CBF and concomitantly spare noncholinergic neurons with similar morphology and projections, 192 IgG-saporin can be used to produce a selective model of CBF deficit in the rat.

Differential roles of TLR2 and TLR4 in acute focal cerebral ischemia/reperfusion injury in mice.

Recent studies have shown that Toll-like receptors (TLRs) are involved in cerebral ischemia/reperfusion (I/R) injury. This study was to investigate the role of TLR2 and TLR4 in acute focal cerebral I/R injury. Cerebral infarct size, neurological function and mortality were evaluated. NFsmall ka, CyrillicB binding activity, phosphorylation of Ismall ka, CyrillicBalpha, Akt and ERK1/2 were examined in ischemic cerebral tissue by EMSA and Western blots. Compared to wild type (WT) mice, in TLR4 knockout (TLR4KO) mice, brain infarct size was decreased (2.6+/-1.18% vs 11.6+/-1.97% of whole cerebral volume, p<0.05) and neurological function was maintained (7.3+/-0.79 vs 4.7+/-0.68, p<0.05). However, compared to TLR4KO mice, TLR2 knockout (TLR2KO) mice showed higher mortality (38.2% vs 13.0%, p<0.05), decreased neurological function (2.9+/-0.53 vs 7.3+/-0.79, p<0.05) and increased brain infarct size (19.1+/-1.33% vs 2.6+/-1.18%, p<0.05). NFsmall ka, CyrillicB activation and Ismall ka, CyrillicBalpha phosphorylation were attenuated in TLR4KO mice (1.09+/-0.02 and 1.2+/-0.04) compared to TLR2KO mice (1.31+/-0.02 and 2.2+/-0.32) after cerebral ischemia. Compared to TLR4KO mice, in TLR2KO mice, the phosphorylation of Akt (0.2+/-0.03 vs 0.9+/-0.16, p<0.05) and ERK1/2 (0.8+/-0.06 vs 1.3+/-0.17) evoked by cerebral I/R was attenuated. The present study demonstrates that TLR2 and TLR4 play differential roles in acute cerebral I/R injury. Specifically, TLR4 contributes to cerebral I/R injury, while TLR2 appears to be neuroprotective by enhancing the activation of protective signaling in response to cerebral I/R.

The effects of immunolesions of nerve growth factor-receptive neurons by 192 IgG-saporin on sleep.

Low-affinity nerve growth factor (NGF) receptors are present on the cholinergic neurons of the basal forebrain. We studied the effects of 192 IgG-saporin, a specific immunotoxin for the NGF receptor-positive, cholinergic basal forebrain neurons, on sleep, the power spectrum of the electroencephalogram (EEG), and body temperature. After 3 d baseline recordings, 12 male rats were injected intracerebroventricularly with 4 micrograms 192 IgG-saporin. EEG, motor activity, and brain temperature were recorded for 23 h on the first, third, fifth, and seventh day after the treatment. 192 IgG-saporin did not affect the total daily amounts but altered the circadian distribution of sleep. On days 1 and 3 after the injection of the immunotoxin, the amount of non-rapid-eye-movement sleep (NREMS) and rapid-eye-movement sleep (REMS) increased during the dark period, whereas during the light both NREMS and REMS decreased. On day 5, these changes were less pronounced and sleep completely returned to the baseline by day 7. The EEG was suppressed in each frequency band and each vigilance state, and, in contrast to sleep, these changes in EEG persisted for 7 days. Brain temperature was decreased from day 3. These results suggest that NGF receptor-positive, cholinergic basal forebrain neurons are not necessary for the maintenance of total sleep time but contribute to the generation of normal EEG and the maintenance of brain temperature.

Resuscitation from severe hemorrhagic shock after traumatic brain injury using saline, shed blood, or a blood substitute

The original purpose of this study was to compare initial resuscitation of hemorrhagic hypotension after traumatic brain injury (TBI) with saline and shed blood. Based on those results, the protocol was modified and saline was compared to a blood substitute, diaspirin cross-linked hemoglobin (DCLHb). Two series of experiments were performed in anesthetized and mechanically ventilated (FiO2 = 0.4) pigs (35–45 kg). In Series 1, fluid percussion TBI (6–8 ATM) was followed by a 30% hemorrhage. At 120 min post-TBI, initial resuscitation consisted of either shed blood (n = 7) or a bolus of 3× shed blood volume as saline (n = 13). Saline supplements were then administered to all pigs to maintain a systolic arterial blood pressure (SAP) of >100 mmHg and a heart rate (HR) of <110 beats/min. In Series 2, TBI (4–5 ATM) was followed by a 35% hemorrhage. At 60 min post-TBI, initial resuscitation consisted of either 500 mL of DCLHb (n = 6) or 500 mL of saline (n = 5). This was followed by saline supplements to all pigs to maintain a SAP of >100 mmHg and a HR of <110 beats/min. In Series 1, most systemic markers of resuscitation (e.g., SAP, HR, cardiac output, filling pressures, lactate, etc.) were normalized, but there were 0/7 vs. 5/13 deaths within 5 h (P = 0.058) with blood vs. saline. At constant arterial O2 saturation (SaO2), mixed venous O2 saturation (SvO2), cerebral perfusion pressure (CPP), and cerebral venous O2 saturation (ScvO2) were all higher, intracranial pressure (ICP) was lower, and CO2 reactivity was preserved with blood vs. saline (all P < 0.05). In Series 2, SAP, ICP, CPP, and lactate were higher with DCLHb vs. saline (all P < 0.05). Cardiac output was lower even though filling pressure was markedly elevated with DCLHb vs. saline (both P < 0.05). Neither SvO2 nor cerebrovascular CO2 reactivity were improved, and ScvO2 was lower with DCLHb vs. saline (P < 0.05). All survived at least 72 h with neuropathologic changes that included sub-arachnoid hemorrhage, midline cerebellar necrosis, and diffuse axonal injury. These changes were similar with DCLHb vs. saline. Thus, whole blood was more effective than saline for resuscitation of TBI, whereas DCLHb was no more, and according to many variables, less effective than saline resuscitation. These experimental results are comparable to those in a recent multicenter trial using DCLHb for the treatment of severe traumatic shock. Further investigations in similar experimental models might provide some plausible explanations why DCLHb unexpectedly increased mortality in patients.