Streptozotocin diabetes results in increased responsiveness of adipocyte lipolysis to glucagon

AbstractAdipocytes from streptozotocin-diabetic rats are ∼50-times more sensitive to the lipolytic action of glucagon. This change is only perceived in the presence of a small quantity of adenosine deaminase which itself has little effect on basal lipolysis. Insulin treatment restores glucagon sensitivity to normal

The Use of Antisense-Mediated Inhibition to Delineate The Role of Inflammatory Agents in The Pathophysiology of Spinal Cord Injury

Injuries to the central nervous system (CNS) usually lead to a potent and acute inflammatory response[1]. During this period, glia and immune cells respond to chemical cues associated with the debris of lysed neurons, disrupted axons, and a broken blood-brain-barrier by releasing a battery of cytokines including tumor necrosis factor-α (TNF-α) and, interleukin-β (IL-1β) as well as reactive oxygen species such as nitric oxide (NO-)[2]. The secretion of these factors may be primarily responsible for secondary damage to surrounding uninjured tissue that potentiates the initial injury[3]. Antisense oligonucleotides (ASOs) are designed to hybridize to specific regions of specific mRNAs. Hybridization of the oligonucleotide to the mRNA then interferes with the normal processing of that mRNA at the ribosome or targets the RNA duplex for cleavage by the RNA digestive enzyme, ribonuclease H, resulting in greatly reduced expression of the coded protein. This effectively reduces the amount of corresponding translated protein product and experiments can be designed to examine the requirement of particular inflammatory agents in eliciting specific deleterious responses after injury, e.g., cell death

Role of nitric oxide in traumatic brain injury in the rat

Object. Although nitric oxide (NO) has been shown to play an important role in the pathophysiological process of cerebral ischemia, its contribution to the pathogenesis of traumatic brain injury (TBI) remains to be clarified. The authors investigated alterations in constitutive nitric oxide synthase (NOS) activity after TBI and the histopathological response to pharmacological manipulations of NO. Methods. Male Sprague—Dawley rats underwent moderate (1.7–2.2 atm) parasagittal fluid-percussion brain injury. Constitutive NOS activity significantly increased within the ipsilateral parietal cerebral cortex, which is the site of histopathological vulnerability, 5 minutes after TBI occurred (234.5 ± 60.2% of contralateral value [mean ± standard error of the mean {SEM}], p < 0.05), returned to control values by 30 minutes (114.1 ± 17.4%), and was reduced at 1 day after TBI (50.5 ± 13.1%, p < 0.01). The reduction in constitutive NOS activity remained for up to 7 days after TBI (31.8 ± 6.0% at 3 days, p < 0.05; 20.1 ± 12.7% at 7 days, p < 0.01). Pretreatment with 3-bromo-7-nitroindazole (7-NI ) (25 mg/kg), a relatively specific inhibitor of neuronal NOS, significantly decreased contusion volume (1.27 ± 0.17 mm3 [mean ± SEM], p < 0.05) compared with that of control (2.52 ± 0.35 mm3). However, posttreatment with 7-NI or pre- or posttreatment with nitro-l-arginine-methyl ester (l-NAME) (15 mg/kg), a nonspecific inhibitor of NOS, did not affect the contusion volume compared with that of control animals (1.87 ± 0.46 mm3, 2.13 ± 0.43 mm3, and 2.18 ± 0.53 mm3, respectively). Posttreatment with l-arginine (1.1 ± 0.3 mm3, p < 0.05), but not 3-morpholino-sydnonimine (SIN-1) (2.48 ± 0.37 mm3), significantly reduced the contusion volume compared with that of control animals. Conclusions. These data indicate that constitutive NOS activity is affected after moderate parasagittal fluid percussion brain injury in a time-dependent manner. Inhibition of activated neuronal NOS and/or enhanced endothelial NOS activation may represent a potential therapeutic strategy for the treatment of TBI

Importance of Posttraumatic Hypothermia and Hyperthermia on the Inflammatory Response after Fluid Percussion Brain Injury: Biochemical and Immunocytochemical Studies

The purpose of this study was to investigate: 1) the temporal and regional profile of polymorphonuclear leukocyte (PMNL) infiltration after moderate traumatic brain injury using the parasagittal fluid percussion model and 2) the effects of posttraumatic hypothermia (30°C) and hyperthermia (39°C) on the acute and subacute inflammatory response. We hypothesized that posttraumatic hypothermia would reduce the degree of PMNL accumulation whereas hyperthermia would exacerbate this response to injury. In the first series of experiments we quantitated the temporal profile of altered myeloperoxidase activity under normothermic (37°C) conditions (n = 20). The rats were allowed to survive for 3 hours, 24 hours, 3 days, or 7 days after trauma, and brains were dissected into cortical and subcortical regions ipsilateral and contralateral to injury. Additional animals were perfused and fixed for the immunocytochemical visualization of myeloperoxidase (n = 15). In the second series of experiments, rats (n = 25) were killed 3 hours or 3 days after the 3-hour monitoring period of normothermia (36.5°C), hypothermia (30°C), or hyperthemia (39°C) (n = 4 to 5 per group), and myeloperoxidase activity was again quantitated. In normothermic rats, the enzymatic activity of myeloperoxidase was significantly increased ( P < 0.05) at 3 hours within the anterior cortical segment (213.97 ± 56.2 versus control 65.5 ± 52.3 U/g of wet tissue; mean ± SD) and posterior (injured) cortical and subcortical segments compared to shamoperated rats (305.76 ± 27.8 and 258.67 ± 101.4 U/g of wet tissue versus control 62.8 ± 24.8 and 37.28 ± 35.6 U/g of wet tissue; P < 0.0001, P < 0.05, respectively). At 24 hours and 7-days after trauma only the posterior cortical region ( P < 0.005, P < 0.05, respectively) exhibited increased myeloperoxidase activity. However, 3 days after trauma, myeloperoxidase activity was also significantly increased within the anterior cortical segment ( P < 0.05) and in posterior cortical and subcortical regions compared to sham-operated cortex ( P < 0.0001, P < 0.05, respectively). Immunocytochemical analysis of myeloperoxidase reactivity at 3 hours, 24 hours, 3- and 7-days demonstrated large numbers of immunoreactive leukocytes within and associated with blood vessels, damaged tissues, and subarachnoid spaces. Posttraumatic hypothermia and hyperthermia had significant effects on myeloperoxidase activity at both 3 hours and 3 days after traumatic brain injury. Posttraumatic hypothermia reduced myeloperoxidase activity in the injured and noninjured cortical and subcortical segments compared to normothermic values ( P < 0.05). In contrast, posttraumatic hyperthermia significantly elevated myeloperoxidase activity in the posterior cortical region compared to normothermic values at both 3 hours and 3 days (473.5 ± 258.4 and 100.11 ± 27.58 U/g of wet tissue, respectively, P < 0.05 versus controls). These results indicate that posttraumatic hypothermia decreases early and more prolonged myeloperoxidase activation whereas hyperthermia increases myeloperoxidase activity. Temperature-dependent alterations in PMNL accumulation appear to be a potential mechanism by which posttraumatic temperature manipulations may influence traumatic outcome

Effects of Moderate Hypothermia on Constitutive and Inducible Nitric Oxide Synthase Activities After Traumatic Brain Injury in the Rat

: We investigated the effects of therapeutic hypothermia (30°C) on alterations in constitutive (cNOS) and inducible (iNOS) nitric oxide synthase activities following traumatic brain injury (TBI). Male Sprague–Dawley rats were anesthetized with 0.5% halothane and underwent moderate (1.8–2.2 atm) parasagittal fluid‐percussion (F‐P) brain injury. In normothermic rats (37°C) the enzymatic activity of cNOS was significantly increased at 5 min within the injured cerebral cortex compared with contralateral values (286.5 ± 68.9% of contralateral value; mean ± SEM). This rise in nitric oxide synthase activity was significantly reduced with pretraumatic hypothermia (138.8 ± 17% of contralateral value; p < 0.05). At 3 and 7 days after normothermic TBI the enzymatic activity of cNOS was decreased significantly (30 ± 8.4 and 28.6 ± 20.9% of contralateral value, respectively; p < 0.05). However, immediate posttraumatic hypothermia (3 h at 30°C) preserved cNOS activity at 3 and 7 days (69.5 ± 23.3 and 78.6 ± 7.6% of contralateral value, respectively; mean ± SEM; p < 0.05). Posttraumatic hypothermia also significantly reduced iNOS activity at 7 days compared with normothermic rats (0.021 ± 0.06 and 0.23 ± 0.06 pmol/mg of protein/min, respectively; p < 0.05). The present results indicate that hypothermia (a) decreases early cNOS activation after TBI, (b) preserves cNOS activity at later periods, and (c) prevents the delayed induction of iNOS. Temperature‐dependent alterations in cNOS and iNOS enzymatic activities may participate in the neuroprotective effect of hypothermia in this TBI model

Temporal and segmental distribution of constitutive and inducible nitric oxide synthases after traumatic spinal cord injury: effect of aminoguanidine treatment

Nitric oxide (NO) has been shown to play an important role in the pathophysiology of traumatic brain injury (TBI) and cerebral ischemia. However, its contribution to the pathogenesis of traumatic spinal cord injury (SCI) remains to be clarified. This study determined the time course of constitutive and inducible nitric oxide synthases (cNOS and iNOS, respectively) after SCI. Rats underwent moderate SCI at T10 using the NYU impactor device and were allowed to survive for 3, 6, or 24 h and 3 days after SCI (n = 5 in each group). For the determination of enzymatic activities, spinal cords were dissected into five segments, including levels rostral and caudal (remote) to the injury site. Other rats were perfusion fixed for the immunohistochemical localization of iNOS protein levels. cNOS activity was significantly decreased at 3 and 6 h within the traumatized T10 segment and at 3, 6, and 24 h at the rostral (T9) level (p < 0.05). Rostral (T8) and caudal (T11, T12) to the injury site cNOS activity was also decreased at 3 h after injury (p < 0.05). However, cNOS activity returned to control levels within 6 h at T8, T11 and T12 and at one day at T10 and T9 segments. iNOS enzymatic activity was elevated at all time points tested (p < 0.05), with the most robust increase observed at 24 h. Immunostaining for iNOS at 24 h revealed that a significant cellular source of iNOS protein appeared to be invading polymorphonuclear leukocytes (PMNLs). To assess the functional consequences of iNOS inhibition, aminoguanidine treatment was initiated 5 min after SCI and rats tested using the BBB open field locomotor score. Treated rats demonstrated significantly improved hindlimb function up to 7 weeks after SCI. Histopathological analysis of contusion volume showed that aminoguanidine treatment decreased lesion volume by 37% (p < 0.05). In conclusion, these results indicate that (1) cNOS and iNOS activities are regionally and temporally affected after moderate SCI, (2) the early accumulation of PMNLs are a potentially significant source of NO-induced cytotoxic products, and (3) acute aminoguanidine treatment significantly improves functional and histopathological outcome after SCI

Tumor necrosis factor alpha expression and protein levels after fluid percussion injury in rats: the effect of injury severity and brain temperature

Tumor necrosis factor alpha (TNFalpha) is elevated in some models of traumatic brain injury (TBI). However, it is unclear how TNFalpha messenger ribonucleic acid (mRNA) expression and protein levels are affected by injury severity and posttraumatic temperature modification. This study determined the regional and temporal profile of TNFalpha levels after moderate and severe TBI and assessed the effects of posttraumatic hypothermia or hyperthermia on this proinflammatory cytokine. Adult male Sprague-Dawley rats were subjected to sham procedures (no injury), moderate fluid-percussion TBI (1.8-2.2 atm), or severe fluid-percussion TBI (2.4-2.6 atm). After 1 to 72 hours of survival, animals were killed, and brain samples, cerebrospinal fluid, and serum were harvested for enzyme-linked immunosorbent assay quantification of TNFalpha levels. In a subsequent study, a 3-hour period of posttraumatic hypothermia (33 degrees C) or hyperthermia (39.5 degrees C) was applied, followed by immediate killing and cytokine assay. Another group was subjected to moderate TBI (1.8-2.2 atm), followed by killing at 15 minutes or at 1, 3, or 24 hours for TNFalpha reverse transcriptase-polymerase chain reaction analysis. A significant increase in TNFalpha mRNA and protein levels in cellular lysates of injured cortex and ipsilateral hippocampus was noted by 1 hour after TBI; it was sustained to 3 hours, followed by a rapid decline. Increased injury severity was associated with increased protein levels at remote injury sites and in the injured cerebral cortex at 72 hours. Posttraumatic hypothermia significantly reduced TNFalpha mRNA expression in the hippocampus compared with that in normothermic rats. In contrast, no temperature effects on TNFalpha protein levels were documented. Rapid and marked increase in TNFalpha mRNA expression and protein levels follows moderate and severe TBI. Injury severity and posttraumatic temperature play a modest but significant role on TNFalpha expression and protein levels. These findings suggest that the effects of posttraumatic temperature on histopathological and behavioral outcome primarily may involve secondary mediators that do not operate directly through their effect on TNFalpha