Effects of Mild Closed-Head Injury and Subanesthetic Ketamine Infusion on Microglia, Axonal Injury, and Synaptic Density in Sprague-Dawley Rats.

Mild traumatic brain injury (mTBI) affects millions of people in the U.S. Approximately 20-30% of those individuals develop adverse symptoms lasting at least 3 months. In a rat mTBI study, the closed-head impact model of engineered rotational acceleration (CHIMERA) produced significant axonal injury in the optic tract (OT), indicating white-matter damage. Because retinal ganglion cells project to the lateral geniculate nucleus (LGN) in the thalamus through the OT, we hypothesized that synaptic density may be reduced in the LGN of rats following CHIMERA injury. A modified SEQUIN (synaptic evaluation and quantification by imaging nanostructure) method, combined with immunofluorescent double-labeling of pre-synaptic (synapsin) and post-synaptic (PSD-95) markers, was used to quantify synaptic density in the LGN. Microglial activation at the CHIMERA injury site was determined using Iba-1 immunohistochemistry. Additionally, the effects of ketamine, a potential neuroprotective drug, were evaluated in CHIMERA-induced mTBI. A single-session repetitive (ssr-) CHIMERA (3 impacts, 1.5 joule/impact) produced mild effects on microglial activation at the injury site, which was significantly enhanced by post-injury intravenous ketamine (10 mg/kg) infusion. However, ssr-CHIMERA did not alter synaptic density in the LGN, although ketamine produced a trend of reduction in synaptic density at post-injury day 4. Further research is necessary to characterize the effects of ssr-CHIMERA and subanesthetic doses of intravenous ketamine on different brain regions and multiple time points post-injury. The current study demonstrates the utility of the ssr-CHIMERA as a rodent model of mTBI, which researchers can use to identify biological mechanisms of mTBI and to develop improved treatment strategies for individuals suffering from head trauma.

Effects of a Subanesthetic Ketamine Infusion on Inflammatory and Behavioral Outcomes after Closed Head Injury in Rats.

Traumatic brain injury (TBI) affects millions of people annually, and most cases are classified as mild TBI (mTBI). Ketamine is a potent trauma analgesic and anesthetic with anti-inflammatory properties. However, ketamine's effects on post-mTBI outcomes are not well characterized. For the current study, we used the Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA), which replicates the biomechanics of a closed-head impact with resulting free head movement. Adult male Sprague-Dawley rats sustained a single-session, repeated-impacts CHIMERA injury. An hour after the injury, rats received an intravenous ketamine infusion (0, 10, or 20 mg/kg, 2 h period), during which locomotor activity was monitored. Catheter blood samples were collected at 1, 3, 5, and 24 h after the CHIMERA injury for plasma cytokine assays. Behavioral assays were conducted on post-injury days (PID) 1 to 4 and included rotarod, locomotor activity, acoustic startle reflex (ASR), and pre-pulse inhibition (PPI). Brain tissue samples were collected at PID 4 and processed for GFAP (astrocytes), Iba-1 (microglia), and silver staining (axonal injury). Ketamine dose-dependently altered locomotor activity during the infusion and reduced KC/GRO, TNF-α, and IL-1ß levels after the infusion. CHIMERA produced a delayed deficit in rotarod performance (PID 3) and significant axonal damage in the optic tract (PID 4), without significant changes in other behavioral or histological measures. Notably, subanesthetic doses of intravenous ketamine infusion after mTBI did not produce adverse effects on behavioral outcomes in PID 1-4 or neuroinflammation on PID 4. A further study is warranted to thoroughly investigate beneficial effects of IV ketamine on mTBI given multi-modal properties of ketamine in traumatic injury and stress.

Loss of Consciousness and Righting Reflex Following Traumatic Brain Injury: Predictors of Post-Injury Symptom Development (A Narrative Review).

Identifying predictors for individuals vulnerable to the adverse effects of traumatic brain injury (TBI) remains an ongoing research pursuit. This is especially important for patients with mild TBI (mTBI), whose condition is often overlooked. TBI severity in humans is determined by several criteria, including the duration of loss of consciousness (LOC): LOC < 30 min for mTBI and LOC > 30 min for moderate-to-severe TBI. However, in experimental TBI models, there is no standard guideline for assessing the severity of TBI. One commonly used metric is the loss of righting reflex (LRR), a rodent analogue of LOC. However, LRR is highly variable across studies and rodents, making strict numeric cutoffs difficult to define. Instead, LRR may best be used as predictor of symptom development and severity. This review summarizes the current knowledge on the associations between LOC and outcomes after mTBI in humans and between LRR and outcomes after experimental TBI in rodents. In clinical literature, LOC following mTBI is associated with various adverse outcome measures, such as cognitive and memory deficits; psychiatric disorders; physical symptoms; and brain abnormalities associated with the aforementioned impairments. In preclinical studies, longer LRR following TBI is associated with greater motor and sensorimotor impairments; cognitive and memory impairments; peripheral and neuropathology; and physiologic abnormalities. Because of the similarities in associations, LRR in experimental TBI models may serve as a useful proxy for LOC to contribute to the ongoing development of evidence-based personalized treatment strategies for patients sustaining head trauma. Analysis of highly symptomatic rodents may shed light on the biological underpinnings of symptom development after rodent TBI, which may translate to therapeutic targets for mTBI in humans.

Effects of an intravenous ketamine infusion on inflammatory cytokine levels in male and female Sprague-Dawley rats.

BACKGROUND: Ketamine, a multimodal dissociative anesthetic drug, is widely used as an analgesic following traumatic injury. Although ketamine may produce anti-inflammatory effects when administered after injury, the immunomodulatory properties of intravenous (IV) ketamine in a non-inflammatory condition are unclear. In addition, most preclinical studies use an intraperitoneal (IP) injection of ketamine, which limits its clinical translation as patients usually receive an IV ketamine infusion after injury. METHODS: Here, we administered sub-anesthetic doses of a single IV ketamine infusion (0, 10, or 40 mg/kg) to male and female Sprague-Dawley rats over a 2-h period. We collected blood samples at 2- and 4-h post-ketamine infusion to determine plasma inflammatory cytokine levels using multiplex immunoassays. RESULTS: The 10 mg/kg ketamine infusion reduced spontaneous locomotor activity in male and female rats, while the 40 mg/kg infusion stimulated activity in female, but not male, rats. The IV ketamine infusion produced dose-dependent and sex-specific effects on plasma inflammatory cytokine levels. A ketamine infusion reduced KC/GRO and tumor necrosis factor alpha (TNF-α) levels in both male and female rats, interleukin-6 (IL-6) levels in female rats, and interleukin-10 (IL-10) levels in male rats. However, most cytokine levels returned to control levels at 4-h post-infusion, except for IL-6 levels in male rats and TNF-α levels in female rats, indicating a different trajectory of certain cytokine changes over time following ketamine administration. CONCLUSIONS: The current findings suggest that sub-anesthetic doses of an IV ketamine infusion may produce sex-related differences in the effects on peripheral inflammatory markers in rodents, and further research is warranted to determine potential therapeutic effects of an IV ketamine infusion in an inflammatory condition.

Enhanced Fear Memories and Altered Brain Glucose Metabolism (18F-FDG-PET) following Subanesthetic Intravenous Ketamine Infusion in Female Sprague-Dawley Rats.

Although women and men are equally likely to receive ketamine following traumatic injury, little is known regarding sex-related differences in the impact of ketamine on traumatic memory. We previously reported that subanesthetic doses of an intravenous (IV) ketamine infusion following fear conditioning impaired fear extinction and altered regional brain glucose metabolism (BGluM) in male rats. Here, we investigated the effects of IV ketamine infusion on fear memory, stress hormone levels, and BGluM in female rats. Adult female Sprague-Dawley rats received a single IV ketamine infusion (0, 2, 10, or 20 mg/kg, over a 2-h period) following auditory fear conditioning (three pairings of tone and footshock). Levels of plasma stress hormones, corticosterone (CORT) and progesterone, were measured after the ketamine infusion. Two days after ketamine infusion, fear memory retrieval, extinction, and renewal were tested over a three-day period. The effects of IV ketamine infusion on BGluM were determined using 18F-fluoro-deoxyglucose positron emission tomography (18F-FDG-PET) and computed tomography (CT). The 2 and 10 mg/kg ketamine infusions reduced locomotor activity, while 20 mg/kg infusion produced reduction (first hour) followed by stimulation (second hour) of activity. The 10 and 20 mg/kg ketamine infusions significantly elevated plasma CORT and progesterone levels. All three doses enhanced fear memory retrieval, impaired fear extinction, and enhanced cued fear renewal in female rats. Ketamine infusion produced dose-dependent effects on BGluM in fear- and stress-sensitive brain regions of female rats. The current findings indicate that subanesthetic doses of IV ketamine produce robust effects on the hypothalamic-pituitary-adrenal (HPA) axis and brain energy utilization that may contribute to enhanced fear memory observed in female rats.

Effects of subanesthetic intravenous ketamine infusion on neuroplasticity-related proteins in male and female Sprague-Dawley rats.

Although ketamine, a multimodal dissociative anesthetic, is frequently used for analgesia and treatment-resistant major depression, molecular mechanisms of ketamine remain unclear. Specifically, differences in the effects of ketamine on neuroplasticity-related proteins in the brains of males and females need further investigation. In the current study, adult male and female Sprague-Dawley rats with an indwelling jugular venous catheter received an intravenous ketamine infusion (0, 10, or 40 mg/kg, 2-h), starting with a 2 mg/kg bolus for ketamine groups. Spontaneous locomotor activity was monitored by infrared photobeams during the infusion. Two hours after the infusion, brain tissue was dissected to obtain the medial prefrontal cortex (mPFC), hippocampus including the CA1, CA3, and dentate gyrus, and amygdala followed by Western blot analyses of a transcription factor (c-Fos), brain-derived neurotrophic factor (BDNF), and phosphorylated extracellular signal-regulated kinase (pERK). The 10 mg/kg ketamine infusion suppressed locomotor activity in male and female rats while the 40 mg/kg infusion stimulated activity only in female rats. In the mPFC, 10 mg/kg ketamine reduced pERK levels in male rats while 40 mg/kg ketamine increased c-Fos levels in male and female rats. Female rats in proestrus/estrus phases showed greater ketamine-induced c-Fos elevation as compared to those in diestrus phase. In the amygdala, 10 and 40 mg/kg ketamine increased c-Fos levels in female, but not male, rats. In the hippocampus, 10 mg/kg ketamine reduced BDNF levels in male, but not female, rats. Taken together, the current data suggest that subanesthetic doses of intravenous ketamine infusions produce differences in neuroplasticity-related proteins in the brains of male and female rats.

Effects of Ketamine on Rodent Fear Memory.

Ketamine, a multimodal anesthetic drug, has become increasingly popular in the treatment of pain following traumatic injury as well as treatment-resistant major depressive disorders. However, the psychological impact of this dissociative medication on the development of stress-related disorders such as post-traumatic stress disorder (PTSD) remains controversial. To address these concerns, preclinical studies have investigated the effects of ketamine administration on fear memory and stress-related behaviors in laboratory animals. Despite a well-documented line of research examining the effects of ketamine on fear memory, there is a lack of literature reviews on this important topic. Therefore, this review article summarizes the current preclinical literature on ketamine and fear memory with a particular emphasis on the route, dose, and timing of ketamine administration in rodent fear conditioning studies. Additionally, this review describes the molecular mechanisms by which ketamine may impact fear memory and stress-related behaviors. Overall, findings from previous studies are inconsistent in that fear memory may be increased, decreased, or unaltered following ketamine administration in rodents. These conflicting results can be explained by factors such as the route, dose, and timing of ketamine administration; the interaction between ketamine and stress; and individual variability in the rodent response to ketamine. This review also recommends that future preclinical studies utilize a clinically relevant route of administration and account for biological sex differences to improve translation between preclinical and clinical investigations.

Sex-related differences in intravenous ketamine effects on dissociative stereotypy and antinociception in male and female rats.

Ketamine, a multimodal dissociative anesthetic drug, is widely used to treat various conditions including acute pain and treatment-resistant depression. We previously reported that subanesthetic doses of intravenous (i.v.) ketamine produced transient dissociative stereotypy and antinociception in male rats. However, sex-related differences in the effects of i.v. ketamine on these measures are not well characterized. Adult male and female Sprague-Dawley rats (10 weeks old) received an i.v. bolus saline or ketamine (2 and 5 mg/kg), and dissociative stereotypy (head weaving, ataxia, and circling) and natural behaviors (horizontal activity, rearing, and grooming) were quantified over a 10-min period. Ten minutes after the behavioral observation, antinociception was measured using a tail flick test. The i.v. ketamine administration increased head weaving, ataxia, circling, and horizontal activity while decreasing rearing and grooming behaviors in male and female rats. Following 5 mg/kg ketamine administration, ataxia was greater in female rats, while head weaving was greater in male rats. Among the female rats, head weaving was greater in the low estrogen group (diestrus phase) as compared to the high estrogen group (proestrus/estrus phase). Ketamine doses (2 and 5 mg/kg) produced antinociception in male and female rats, and female rats were more sensitive to the antinociceptive effects of 2 mg/kg ketamine. The current findings suggest that i.v. ketamine administration, a clinically relevant route of administration, may produce sex-related differences in dissociative behaviors and analgesia between males and females.

Association between intravenous ketamine-induced stress hormone levels and long-term fear memory renewal in Sprague-Dawley rats.

Ketamine is a multimodal dissociative anesthetic and analgesic that is widely used after traumatic injury. We previously reported that an analgesic dose of intravenous (IV) ketamine infusion (10 mg/kg, 2-h) after fear conditioning enhanced short-term fear memory in rats. Here, we investigated the effects of the same dose of an IV ketamine infusion on plasma stress hormone levels and long-term fear memory in rats. Adult male Sprague-Dawley rats (9-week-old with an average weight of 308 g upon arrival) received a ketamine infusion (0 or 10 mg/kg, 2-h) immediately after auditory fear conditioning (three auditory tone and footshock [0.6 mA, 1-s] pairings) on Day 0. After the infusion, a blood sample was collected from a jugular vein catheter for corticosterone and progesterone assays, and each animal was tested on tail flick to measure thermal antinociception. One week later, animals were tested on fear extinction acquisition (Day 7), fear extinction retrieval (Day 8), and fear renewal (Day 9). The IV ketamine infusion, compared to the saline infusion, reduced locomotor activity (sedation), increased tail flick latency (antinociception), and elevated plasma corticosterone and progesterone levels. The ketamine infusion did not alter long-term fear memory extinction or fear renewal. However, elevated corticosterone and progesterone levels resulting from the ketamine infusion were correlated with sedation, antinociception, and long-term fear memory renewal. These results suggest that individual differences in sensitivity to acute ketamine may predict vulnerability to develop fear-related disorders.

Effects of subanesthetic intravenous ketamine infusion on neuroplasticity-related proteins in the prefrontal cortex, amygdala, and hippocampus of Sprague-Dawley rats.

Ketamine, a multimodal dissociative anesthetic, is a powerful analgesic administered following trauma due to its hemodynamic and respiratory stability. However, ketamine can cause hallucination and dissociation which may adversely impact traumatic memory after an injury. The effects of ketamine on proteins implicated in neural plasticity are unclear due to different doses, routes, and timing of drug administration in previous studies. Here, we investigated the effects of a single intravenous (IV) ketamine infusion on protein levels in three brain regions of rats. Adult male Sprague-Dawley rats with indwelling IV catheters underwent an auditory fear conditioning (three pairings of tone and mild footshock 0.8 mA, 0.5 s) and received a high dose of IV ketamine (0 or 40 mg/kg/2 h) infusion (Experiment 1). In a follow-up study, animals received a low dose of IV ketamine (0 or 10 mg/kg/2 h) infusion (Experiment 2). Two hours after the infusion, brain tissue from the medial prefrontal cortex (mPFC), hippocampus, and amygdala were collected for western blot analyses. Protein levels of a transcription factor (c-Fos), brain-derived neurotrophic factor (BDNF), and phosphorylated extracellular signal-regulated kinase (pERK) were quantified in these regions. The 40 mg/kg ketamine infusion increased c-Fos levels in the mPFC and amygdala as well as pERK levels in the mPFC and hippocampus. The 10 mg/kg ketamine infusion increased BDNF levels in the amygdala, but decreased pERK levels in the mPFC and hippocampus. These findings suggest that a clinically relevant route of ketamine administration produces dose-dependent and brain region-specific effects on proteins involved in neuroplasticity.

Enhanced fear memories and brain glucose metabolism (18F-FDG-PET) following sub-anesthetic intravenous ketamine infusion in Sprague-Dawley rats.

Ketamine is a multimodal dissociative anesthetic, which provides powerful analgesia for victims with traumatic injury. However, the impact of ketamine administration in the peri-trauma period on the development of post-traumatic stress disorder (PTSD) remains controversial. Moreover, there is a major gap between preclinical and clinical studies because they utilize different doses and routes of ketamine administration. Here, we investigated the effects of sub-anesthetic doses of intravenous (IV) ketamine infusion on fear memory and brain glucose metabolism (BGluM) in rats. Male Sprague-Dawley rats received an IV ketamine infusion (0, 2, 10, and 20 mg/kg, 2 h) or an intraperitoneal (IP) injection (0 and 10 mg/kg) following an auditory fear conditioning (3 pairings of tone and foot shock [0.6 mA, 1 s]) on day 0. Fear memory retrieval, fear extinction, and fear recall were tested on days 2, 3, and 4, respectively. The effects of IV ketamine infusion (0 and 10 mg/kg) on BGluM were measured using 18F-fluoro-deoxyglucose positron emission tomography (FDG-PET) and computed tomography (CT). The IV ketamine infusion dose-dependently enhanced fear memory retrieval, delayed fear extinction, and increased fear recall in rats. The IV ketamine (10 mg/kg) increased BGluM in the hippocampus, amygdala, and hypothalamus, while decreasing it in the cerebellum. On the contrary, a single ketamine injection (10 mg/kg, IP) after fear conditioning facilitated fear memory extinction in rats. The current findings suggest that ketamine may produce differential effects on fear memory depending on the route and duration of ketamine administration.

Dose-response characteristics of intravenous ketamine on dissociative stereotypy, locomotion, sensorimotor gating, and nociception in male Sprague-Dawley rats.

Clinicians administer subanesthetic intravenous (IV) ketamine infusions for treatment of refractory depression, chronic pain, and post-traumatic stress disorder in humans. However, ketamine is administered via the subcutaneous (SC) or intraperitoneal (IP) routes to rodents in most pre-clinical research, which may limit translational application. The present study characterized the dose-response of a subanesthetic IV ketamine bolus (2 and 5mg/kg) and 1-h infusion (5, 10, and 20mg/kg/h) on dissociative stereotypy, locomotion, sensorimotor gating, and thermal nociception in male Sprague-Dawley rats. The secondary aim was to measure ketamine and norketamine plasma concentrations following IV ketamine bolus at 1, 20, and 50min and at the conclusion of the 1-h infusion using liquid chromatography/mass spectrometry. The results showed that ketamine bolus and infusions produced dose-dependent dissociative stereotypy. Bolus (2 and 5mg/kg) and 20mg/kg/h infusion increased locomotor activity while 5mg/kg/h infusion decreased locomotor activity. Both 10 and 20mg/kg/h infusions reduced the acoustic startle reflex, while 5mg/kg bolus and 20mg/kg/h infusion impaired pre-pulse inhibition. Ketamine 5mg/kg bolus and the 10 and 20mg/kg/h infusions induced significant and prolonged antinociception to the hotplate test. Plasma concentrations of ketamine decreased quickly after bolus while norketamine levels increased from 1 to 20min and plateaued from 20 to 50min. The peak ketamine plasma concentrations [ng/ml] were similar between 5mg/kg bolus [4100] vs. 20mg/kg/h infusion [3900], and 2mg/kg bolus [1700] vs. 10mg/kg/h infusion [1500]. These results support the findings from previous ketamine injection studies and further validate the feasibility of administering subanesthetic doses of IV ketamine infusion to rats for neuropharmacological studies.

Prophylactic isopropyl alcohol inhalation and intravenous ondansetron versus ondansetron alone in the prevention of postoperative nausea and vomiting in high-risk patients.

Patients identified as high risk for postoperative nausea and vomiting (PONV) are often treated prophylactically with intravenous (IV) ondansetron and an additional agent. Limited options exist for a second agent with no adverse effects. The purpose of this investigation was to determine if combining the prophylactic inhalation of isopropyl alcohol (IPA) vapors, an agent with no adverse effects, with IV ondansetron would be more effective than IV ondansetron alone in the prevention of PONV in high-risk patients. A total of 76 patients at high risk for PONV were randomized into control (n = 38) and experimental (n = 38) groups. All patients received IV ondansetron before emergence from general anesthesia. In addition, the experimental group inhaled IPA vapors before induction. Severity of PONV was measured using a 0 to 10 verbal numeric rating scale. Other measured variables included time to onset and incidence of PONV, 24-hour composite nausea score, and satisfaction with nausea control. No significant differences in demographics, surgical or anesthesia time, number of risk factors, severity or incidence of PONV, or satisfaction scores were noted. Prophylactic inhalation of IPA vapors in combination with IV ondansetron was no more efficacious than IV ondansetron alone in the prevention of PONV in a high-risk population.