Jesse K. Sulzer

Endocannabinoid Degradation Inhibition Improves Neurobehavioral Function, Blood–Brain Barrier Integrity, and Neuroinflammation following Mild Traumatic Brain Injury

Traumatic brain injury (TBI) is an increasingly frequent and poorly understood condition lacking effective therapeutic strategies. Inflammation and oxidative stress (OS) are critical components of injury, and targeted interventions to reduce their contribution to injury should improve neurobehavioral recovery and outcomes. Recent evidence reveals potential protective, yet short-lived, effects of the endocannabinoids (ECs), 2-arachidonoyl glycerol (2-AG) and N-arachidonoyl-ethanolamine (AEA), on neuroinflammatory and OS processes after TBI. The aim of this study was to determine whether EC degradation inhibition after TBI would improve neurobehavioral recovery by reducing inflammatory and oxidative damage. Adult male Sprague-Dawley rats underwent a 5-mm left lateral craniotomy, and TBI was induced by lateral fluid percussion. TBI produced apnea (17±5 sec) and a delayed righting reflex (479±21 sec). Thirty minutes post-TBI, rats were randomized to receive intraperitoneal injections of vehicle (alcohol, emulphor, and saline; 1:1:18) or a selective inhibitor of 2-AG (JZL184, 16 mg/kg) or AEA (URB597, 0.3 mg/kg) degradation. At 24 h post-TBI, animals showed significant neurological and -behavioral impairment as well as disruption of blood-brain barrier (BBB) integrity. Improved neurological and -behavioral function was observed in JZL184-treated animals. BBB integrity was protected in both JZL184- and URB597-treated animals. No significant differences in ipsilateral cortex messenger RNA expression of interleukin (IL)-1β, IL-6, chemokine (C-C motif) ligand 2, tumor necrosis factor alpha, cyclooxygenase 2 (COX2), or nicotinamide adenine dinucleotide phosphate oxidase (NOX2) and protein expression of COX2 or NOX2 were observed across experimental groups. Astrocyte and microglia activation was significantly increased post-TBI, and treatment with JZL184 or URB597 blocked activation of both cell types. These findings suggest that EC degradation inhibition post-TBI exerts neuroprotective effects. Whether repeated dosing would achieve greater protection remains to be examined.

Alcohol abuse and the injured host: dysregulation of counterregulatory mechanisms review.

ABSTRACT Traumatic injury ranks as the number one cause of death for the younger than 44 years age group and fifth leading cause of death overall (www.nationaltraumainstitute.org/home/trauma_statistics.html). Although improved resuscitation of trauma patients has dramatically reduced immediate mortality from hemorrhagic shock, long-term morbidity and mortality continue to be unacceptably high during the postresuscitation period particularly as a result of impaired host immune responses to subsequent challenges such as surgery or infection. Acute alcohol intoxication (AAI) is a significant risk factor for traumatic injury, with intoxicating blood alcohol levels present in more than 40% of injured patients. Severity of trauma, hemorrhagic shock, and injury is higher in intoxicated individuals than that of sober victims, resulting in higher mortality rates in this patient population. Necessary invasive procedures (surgery, anesthesia) and subsequent challenges (infection) that intoxicated trauma victims are frequently subjected to are additional stresses to an already compromised inflammatory and neuroendocrine milieu and further contribute to their morbidity and mortality. Thus, dissecting the dynamic imbalance produced by AAI during trauma is of critical relevance for a significant proportion of injured victims. This review outlines how AAI at the time of hemorrhagic shock not only prevents adequate responses to fluid resuscitation but also impairs the ability of the host to overcome a secondary infection. Moreover, it discusses the neuroendocrine mechanisms underlying alcohol-induced hemodynamic dysregulation and its relevance to host defense restoration of homeostasis after injury.

Systemic administration of a centrally acting acetylcholinesterase inhibitor improves outcome from hemorrhagic shock during acute alcohol intoxication.

Previously, we have demonstrated that acute alcohol intoxication impairs hemodynamic counter-regulation to hemorrhage in unanesthetized rats, and that this phenomenon is associated with an impaired neuroendocrine response to blood loss. Moreover, we demonstrated that central acetylcholinesterase inhibition restores the hemodynamic and neuroendocrine responses to hemorrhage in alcohol-intoxicated rats. We hypothesized that similar responses could be elicited by systemic administration of physostigmine, an acetylcholinesterase inhibitor that penetrates the blood brain barrier. The relevance of this approach was to establish effectiveness of a more clinically applicable route of drug administration than that used previously. Chronically catheterized adult male Sprague-Dawley rats (250-275 g) were administered a bolus of physostigmine (i.v., 100 &mgr;g/kg) at rest, and in a separate study, simultaneously with Ringers lactate solution after an overnight intragastric infusion of 30% alcohol (∼7 g/kg for 15 h) or 52% isocaloric dextrose and fixed-pressure hemorrhage. I.v. physostigmine administration immediately increased sympathetic outflow via activation of central nicotinic receptors and improved the pressor response to fluid resuscitation in both dextrose controls and alcohol-intoxicated animals. The improved hemodynamic recovery achieved with physostigmine was also associated with attenuation of the rises in the markers of liver and renal damage alanine aminotransferase and blood urea nitrogen in alcohol-intoxicated animals. Additional studies are warranted to determine the effect of central acetylcholinesterase inhibition on tissue injury and survival after severe blood loss, as well as its effects on long-term metabolic and inflammatory responses.

Augmented central nitric oxide production inhibits vasopressin release during hemorrhage in acute alcohol-intoxicated rodents

Acute alcohol intoxication (AAI) attenuates the AVP response to hemorrhage, contributing to impaired hemodynamic counter-regulation. This can be restored by central cholinergic stimulation, implicating disrupted signaling regulating AVP release. AVP is released in response to hemorrhage and hyperosmolality. Studies have demonstrated nitric oxide (NO) to play an inhibitory role on AVP release. AAI has been shown to increase NO content in the paraventricular nucleus. We hypothesized that the attenuated AVP response to hemorrhage during AAI is the result of increased central NO inhibition. In addition, we predicted that the increased NO tone during AAI would impair the AVP response to hyperosmolality. Conscious male Sprague-Dawley rats (300-325 g) received a 15-h intragastric infusion of alcohol (2.5 g/kg + 300 mg·kg(-1)·h(-1)) or dextrose prior to a 60-min fixed-pressure hemorrhage (∼40 mmHg) or 5% hypertonic saline infusion (0.05 ml·kg(-1)·min(-1)). AAI attenuated the AVP response to hemorrhage, which was associated with increased paraventricular NO content. In contrast, AAI did not impair the AVP response to hyperosmolality. This was accompanied by decreased paraventricular NO content. To confirm the role of NO in the alcohol-induced inhibition of AVP release during hemorrhage, the nitric oxide synthase inhibitor, nitro-l-arginine methyl ester (l-NAME; 250 μg/5 μl), was administered centrally prior to hemorrhage. l-NAME did not further increase AVP levels during hemorrhage in dextrose-treated animals; however, it restored the AVP response during AAI. These results indicate that AAI impairs the AVP response to hemorrhage, while not affecting the response to hyperosmolality. Furthermore, these data demonstrate that the attenuated AVP response to hemorrhage is the result of augmented central NO inhibition.

Hypertonic saline resuscitation enhances blood pressure recovery and decreases organ injury following hemorrhage in acute alcohol intoxicated rodents.

BACKGROUND Acute alcohol intoxication (AAI) impairs the hemodynamic and arginine vasopressin (AVP) counter-regulation to hemorrhagic shock (HS) and lactated Ringer’s solution (LR) fluid resuscitation (FR). The mechanism of AAI-induced suppression of AVP release in response to HS involves accentuated nitric oxide (NO) inhibitory tone. In contrast, AAI does not prevent AVP response to increased osmolarity produced by hypertonic saline (HTS) infusion. We hypothesized that FR with HTS during AAI would enhance AVP release by decreasing periventricular nucleus NO inhibitory tone, subsequently improving mean arterial blood pressure (MABP) and organ perfusion. METHODS Male Sprague-Dawley rats received a 15-hour alcohol infusion (2.5 g/kg + 0.3 g/kg/h) or dextrose (DEX) before HS (40 mm Hg × 60 minutes) and FR with HTS (7.5%, 4 ml/kg) or LR (2.4 × blood volume removed). Organ blood flow was determined, and brains were collected for NO content at 2 hours after FR. RESULTS HTS improved MABP recovery in AAI (109 vs. 80 mm Hg) and DEX (114 vs. 83 mm Hg) animals compared with LR. This was associated with higher (>60%) circulating AVP levels at 2 hours after FR compared with those detected in LR animals in both groups. Neither AAI alone nor HS in DEX animals resuscitated with LR altered organ blood flow. In AAI animals, HS and FR with LR reduced blood flow to the liver (72%), small intestine (65%), and large intestine (67%) compared with shams. FR with HTS improved liver (threefold) and small intestine (twofold) blood flow compared with LR in AAI-HS animals. The enhanced MABP response to HTS was prevented by pretreatment with a systemic AVP V1a receptor antagonist. HTS decreased periventricular nucleus NO content in both groups 2 hours after FR. CONCLUSION These results suggest that FR with HTS in AAI results in the removal of central NO inhibition of AVP, restoring AVP levels and improving MABP and organ perfusion in AAI-HS.

DELAYED RESUSCITATION WITH PHYSOSTIGMINE INCREASES END ORGAN DAMAGE IN ALCOHOL INTOXICATED RATS

Previous studies from our laboratory have identified a role for blunted central sympathetic activation in the acute alcohol intoxication (AAI)-induced impairment of the counterregulatory response to hemorrhagic shock (HS). Immediate fluid resuscitation (FR) with acetylcholinesterase inhibitors restores the neuroendocrine and pressor responses to FR in AAI + HS. We hypothesized this intervention would remain beneficial after delay and that restoration of mean arterial blood pressure (MABP) during FR would attenuate organ damage. Male Sprague-Dawley rats received a primed constant alcohol infusion (2.5 g · kg−1 + 0.3 g · kg−1 · h−1 for 15 h) or isocaloric dextrose (DEX) before HS (40 mmHg for 60 min) and FR with lactated Ringers (LR) solution ± physostigmine (PHYS; 100 µg · kg−1) immediately or after a 60-min delay after HS. Immediate LR solution elevated MABP in DEX + HS. Acute alcohol intoxication delayed the initial MABP recovery. Delayed LR solution did not further increase MABP in DEX- or AAI + HS. LR solution + PHYS increased MABP in DEX- and AAI + HS after immediate and delayed FR. No differences were noted in markers of organ dysfunction (alanine aminotransferase [ALT], aspartate aminotransferase, blood urea nitrogen, creatinine) after DEX + HS, and this was unaltered by immediate or delayed LR solution + PHYS. Acute alcohol intoxication + HS increased ALT, which was attenuated by immediate LR solution + PHYS. In contrast, delayed LR solution + PHYS exacerbated tissue injury in AAI + HS, as reflected by increased ALT, aspartate aminotransferase, blood urea nitrogen, creatinine, and liver protein carbonylation over time-matched LR solution. In conclusion, PHYS enhanced blood pressure recovery independent of time of FR and presence of AAI. However, in AAI + HS, delayed LR solution + PHYS accentuated organ damage and dysfunction. These findings suggest that although enhancing the sympathetic response can improve hemodynamic recovery during AAI, it may compromise tissue perfusion and enhance tissue injury.

Sympathetic Modulation of the Host Defense Response to Infectious Challenge during Recovery from Hemorrhage

Background: Trauma/hemorrhage (TxHem) is associated with an immediate pro-inflammatory response that, if exaggerated or prolonged, is thought to contribute to the subsequent immunosuppression that characterizes the period after injury. Previously we have demonstrated that chemical sympathectomy (SNSx) accentuates this immediate pro-inflammatory response to TxHem. These findings suggest that the noradrenergic system plays a critical role in limiting the magnitude of the inflammatory response during TxHem and preserving the integrity of the host defense response to a subsequent infectious challenge during the period after TxHem. Objective: To examine the contribution of tissue norepinephrine to the host defense response to an infectious challenge during recovery from TxHem. Methods: Male Sprague-Dawley rats underwent SNSx (6-hydroxydopamine, i.p. daily for 3 days) prior to vascular catheter implantation. Conscious, unrestrained rats were subjected to traumatic injury (muscle crush) prior to a fixed-pressure hemorrhage (40 mm Hg for 60 min) and fluid resuscitation followed 24 h later by cecal ligation and puncture (CLP). Results: SNSx impaired the hemodynamic and thermoregulatory response to hemorrhage as indicated by decreased basal blood pressure, impaired blood pressure recovery during fluid resuscitation, and greater hypothermia after CLP. Furthermore, SNSx accentuated the TNF-α, IL-1, IL-6, and IL-10 response to TxHem + infection in plasma 6 h after CLP and in peritoneal lavage fluid 24 h after CLP. Conclusion: These results indicate that the integrity of the noradrenergic system is necessary for adequate hemodynamic, thermoregulatory, and inflammatory responses to infection during the period following TxHem.