Ruth V. W. Dimlich

Influence of the sympathetic nervous system as well as trigeminal sensory fibres on rat dural mast cells.

The dura mater has attracted considerable attention as an exquisitely sensitive tissue implicated as playing a role in various cephalalgias including vascular headache. Because of the potential clinical impact of the relationship/interaction of neural elements and mast cells the influence of sensory and autonomic nerve fibres on mast cells of the rat dura mater was studied. The trigeminal or superior cervical ganglion was electrically stimulated and the mast cells were examined. Wholemount supratentorial dural preparations were stained using berberine sulphate and the number of mast cells with intact vs dispersed granules counted. Unilateral stimulation of either ganglia resulted in a statistically significant increase in the percentage of mast cells with dispersed granules ipsilateral to the side of stimulation. These results support our idea that in addition to the trigeminal system the sympathetic nervous system must be considered as playing a role in the oedema pathophysiology of vascular headache.

Myofibrillar proteinase, cathepsin B, and protein breakdown rates in skeletal muscle from septic rats

Muscle catabolism during sepsis is mainly caused by myofibrillar protein breakdown. The mechanism of this metabolic response is not known. We tested the hypothesis that increased protein breakdown in the extensor digitorum longus (EDL) muscle of septic rats is caused by increased activity of the so-called myofibrillar proteinase, which is a nonlysosomal proteolytic enzyme, and cathepsin B, which is a lysosomal proteinase. Sepsis, induced in male Sprague-Dawley rats (50 to 60 g) by cecal ligation and puncture (CLP), resulted in an approximately 50% increase in myofibrillar proteinase activity and an approximately 30% increase in cathepsin B activity. Concomitantly, both total and myofibrillar protein breakdown rates, measured as release of tyrosine and 3-methylhistidine (3-MH), respectively, by incubated EDL muscles, were substantially elevated. Treatment of septic rats with the mast cell degranulating compound 48/80 or the lysosomal protease inhibitor leupeptin significantly reduced myofibrillar proteinase and cathepsin B activities, but did not affect protein breakdown rates. The results suggest that increased protein breakdown in septic skeletal muscle is associated with, but not caused by, myofibrillar proteinase or cathepsin B activity. The data also support the concept of a mast cell origin of the myofibrillar proteinase activity, but do not suggest an obligatory involvement of mast cell proteinase in increased protein degradation during sepsis.

The Fluorescent Staining of Heparin in Mast Cells Using Berberine Sulfate: Compatibility with Paraformaldehyde or o-Phthalaldehyde Induced Fluorescence and Metachromasia

The berberine sulfate technique of Enerback (1974) for the demonstration of heparin was applied to freeze-dried or routinely fixed paraffin embedded sections from various tissues. Sections from tissue which had been freeze-dried were deparaffinized, fixed in Carnoy Formula A for 15 minutes, rinsed in ethanol, hydrated, stained for 20 minutes in a 0.02% aqueous solution of berberine sulfate at a pH of 1, 2, 3, 4, 5 or 6 and rinsed in distilled water at a pH corresponding to the staining solutions. Carnoy Formula A or formalin-fixed tissues were routinely processed and sections were stained as above. Also, sections of freeze-dried tissues which had been deparaffinized and treated with paraformaldehyde or o-phthalaldehyde were hydrated to quench the fluorescence due to serotonin or histamine and restained with aqueous solutions of berberine sulfate (pH 1—6). In sections of tissue at a pH above 4, yellow fluorescence was produced by berberine sulfate in both cartilage and mast cells while in those treated at ...

Postinsult treatment of ischemia-induced cerebral lactic acidosis in the rat

Cerebral ischemic insult is one of the most clinically significant conditions leading to irreversible brain cell damage and death. Animal studies have suggested that lowered intracellular pH due to the severe brain lactic acidosis following ischemia interferes with normal cell structure and function and leads to brain cell necrosis. Therefore, efforts directed to decreasing brain lactate may be beneficial in preventing brain cell damage and death. The goal of our study was to evaluate the effectiveness of postinsult treatment with dichloroacetate (DCA) in controlling increases in brain lactate following partial global ischemia (PGI) in rats. PGI was induced by bilateral carotid artery occlusion and induced hypotension. Animals that received DCA immediately after a 30-minute ischemic insult (n = 5) or 15 minutes after the end of an ischemic insult (n = 5) had cortical lactate levels that were significantly lower (P less than .005) than lactate levels in untreated insulted animals and that were not significantly different than those previously obtained with preinsult DCA treatment in rats subjected to 30 minutes of PGI. Treatment of rats with DCA following PGI may be effective in reducing cortical lactate levels and hence may limit irreversible damage to brain cells following cerebral ischemia.

Mechanisms of ischemic cerebral injury

Normal compensatory mechanisms protect the central nervous system (CNS) from moderate hypoxia and ischemia; however, after more severe ischemia progressive brain hypoperfusion ensues and irreversible damage occurs. Ischemic brain injury remains greatly significant clinically and elucidating the determinants of ischemic neuronal injury and death continues to challenge researchers. Although altered perfusion and decreased energy charge may contribute to the production of irreversible damage, the distribution of lesions seen after insult does not correspond with the degree of ischemic blood flow impairment, nor can neuronal energy deprivation explain the cell damage. Other factors, such as derangements in astrocyte function, calcium homeostasis, free radical metabolism, acid-base regulation and excitatory neurotransmitters also probably mediate ischemic neuronal death. Continued investigation to establish the cellular pathophysiology of cerebral ischemia can guide rational research and therapeutic strategies.

Brain lactate during partial global ischemia and reperfusion: Effect of pretreatment with dichloroacetate in a rat model☆

Elevated cerebral lactate levels following cerebral ischemia have been associated with brain cell damage and death. We previously found that pre- or postischemia treatment with dichloroacetate (DCA), presumably by its activation of brain pyruvate dehydrogenase, effectively lowers cerebral lactate levels in rats subjected to 30 minutes of partial global ischemia (PGI) followed by 30 minutes of recirculation. The goal of the present study was to determine the effects of preischemia DCA treatment on cortical lactate levels during the ischemia period or during early recirculation. Rats (four in each group) received preischemia treatment with DCA and were then subjected to 0, 10, or 30 minutes of PGI or 30 minutes of PGI followed by 15 minutes of recirculation. Cortical lactate levels in pretreated animals were not significantly different from lactate levels of untreated rats at any time during PGI, but were significantly lower than levels in untreated rats at 15 minutes of recirculation (P less than .05, ANOVA). These results suggest that preischemia treatment with DCA does not limit the accumulation of cortical lactate during PGI but may promote its clearance during recirculation following PGI. If reperfusion events influence the degree of brain cell injury, DCA may enhance cell recovery by lower cortical lactate levels in the reperfusion period.

Dichloroacetate treatment of ischemic cerebral lactic acidosis in the fed rat

Despite advances in cardiac resuscitation, ischemic brain injury remains generally untreatable. Animal studies of brain ischemia associate brain lactate levels of more than 18 mumol/g with irreversible neuronal injury. Lowering brain lactate therefore may prevent or minimize ischemic brain necrosis. Earlier studies in our laboratory using fasted rats demonstrated that sodium dichloroacetate (DCA) decreases ischemic brain lactate when given either before or immediately after partial global ischemia (PGI). Other investigators have shown that fed animals have more glucose and generate higher lactate levels by anaerobic metabolism during PGI. We evaluated the ability of DCA to lower brain lactate in fed male Wistar rats subjected to PGI. Four groups (n = 6 each) were studied--PGI and control rats with either placebo or DCA treatment. PGI was induced for 30 minutes by combining bilateral carotid artery occlusion with hemorrhagic hypotension. This was followed by release of carotid occlusion, reinfusion of shed blood, and immediate treatment with either DCA (25 mg/kg, IV) or placebo. Thirty minutes later brains were frozen in situ with liquid nitrogen for extraction and measurement of tissue glucose, glycogen, and lactate. Blood glucose and serum lactate were monitored throughout the experiment. No significant differences were found between the two PGI groups in brain glucose, brain glycogen, or ischemia-induced elevations in blood glucose and serum lactate. However, brain lactate was significantly lower in DCA-treated (12.5 mumol/g) than in untreated (22.8 mumol/g) PGI rats (P less than .001). In addition, all untreated PGI rats had levels of more than 18 mumol/g, and therefore were at high risk for neuronal necrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Dichloroacetate attenuates neuronal damage in a gerbil model of brain ischemia.

Dichloroacetate facilitated a reduction in brain lactate following ischemia in the gerbil. This treatment also improved high-energy metabolite and pyruvate dehydrogenase enzyme recovery. The purpose of this study was to determine the effect of dichloroacetate on ischemia-induced neuronal damage in the hippocampus of the gerbil. In adult male gerbils, carotid arteries were clamped bilaterally for 5 min. After ischemia, each gerbil was graded neurologically and received an ip injection of dichloroacetate (75 or 225 mg/kg) or an equal volume (5 mL/kg) of sodium acetate (66 mg/kg). On the following morning, gerbils received a second injection, and 3 d later were anesthetized and perfused intracardially. Brains were processed, and stained sections were analyzed for neuronal damage. Gerbils treated with 225 mg/kg dichloroacetate exhibited significantly less damage than the untreated group (p=0.05, Dunn’s test). Gerbils with a normal neurologic score evidenced no neuronal damage. Abnormal neurologic scores immediately after ischemia did not correlate with degree of neuronal damage observed 4 d later. These results indicate that neuronal damage is less in gerbils treated after ischemia with an appropriate dose of dichloroacetate. The lack of any histological evidence for an adverse effect of dichloroacetate in the controls supports the safety of this drug in this protocol. Normal neurologic scores immediately after ischemia can be used to identify gerbils mimicking ischemia in this model.

Histamine antagonists in the treatment of shock hyperglycemia in the rat

The present study was undertaken to determine the effect of exogenous histamine and histamine blockers on blood glucose and hepatic glycogen in the rat. Forty-one nonfasted male Sprague-Dawley rats that had been anesthetized with intraperitoneal injections of urethane were injected intravenously (femoral) with histamine (10 mg/kg) five minutes after pretreatment with Ringers solution (control), diphenhydramine (1 mg/kg) (H-1 blocker); metiamide (1 mg/kg) (H-2 blocker); or a combination of these blockers. Mean arterial pressure (carotid), blood glucose, and hepatic glycogen were measured. Within 30 minutes, histamine evoked a significant increase in blood glucose, and a decrease in hepatic glycogen, and a reduction in blood pressure. However, rats treated with the H-2 blocker metiamide or with a combination of H-1 and H-2 blockers did not show as significant a hypotensive response as rats treated with the H-1 blocker diphenhydramine alone. The hyperglycemic-glycogenolytic response to histamine was modified by diphenhydramine as well as by a combination of blockers, but not by metiamide alone. These results suggest that a) the hypotension did not initiate the hyperglycemic and glycogenolytic response; b) the H-2 blocker metiamide has little effect on the hyperglycemic response to exogenous histamine; and c) the H-1 blocker diphenhydramine may have antihyperglycemic properties.

Elevated Blood Glucose after Compound 48/80 Treatment Is Not Related to Hepatic Mast Cell Degranulation in Rats

Abstract Blood glucose, hepatic glycogen, and the histological integrity of hepatic mast cells, were evaluated in anesthetized rats receiving iv injections of 0.125 mg/kg body weight compound 48/80 (a mast cell degranulator) and/or of 0.001 to 10.0 mg/kg body weight lodoxamide tromethamine (an inhibitor of mast cell degranulation). A nonglucogenic dose of lodoxamide, 0.001 mg/kg body weight, prevented dissipation of histochemically demonstrable fluorescence in mast cells (degranulation) without inhibiting compound 48/80-induced hyperglycemia and hepatic glycogenosis. These results suggest that this glucotropic response is independent of compound 48/80-evoked release of mediators such as serotonin from mast cells.