Lateral fluid percussion injury: A rat model of experimental traumatic brain injury.

Traumatic brain injury (TBI) represents one of the leading causes of disability and death worldwide. The annual economic impact of TBI-including direct and indirect costs-is high, particularly impacting low- and middle-income countries. Despite extensive research, a comprehensive understanding of the primary and secondary TBI pathophysiology, followed by the development of promising therapeutic approaches, remains limited. These fundamental caveats in knowledge have motivated the development of various experimental models to explore the molecular mechanisms underpinning the pathogenesis of TBI. In this context, the Lateral Fluid Percussion Injury (LFPI) model produces a brain injury that mimics most of the neurological and systemic aspects observed in human TBI. Moreover, its high reproducibility makes the LFPI model one of the most widely used rodent-based TBI models. In this chapter, we provide a detailed surgical protocol of the LFPI model used to induce TBI in adult Wistar rats. We further highlight the neuroscore test as a valuable tool for the evaluation of TBI-induced sensorimotor consequences and their severity in rats. Lastly, we briefly summarize the current knowledge on the pathological aspects and functional outcomes observed in the LFPI-induced TBI model in rodents.

Cannabidiol Modifies the Glutamate Over-Release in Brain Tissue of Patients and Rats with Epilepsy: A Pilot Study.

Drug-resistant epilepsy (DRE) is associated with high extracellular levels of glutamate. Studies support the idea that cannabidiol (CBD) decreases glutamate over-release. This study focused on investigating whether CBD reduces the evoked glutamate release in cortical synaptic terminals obtained from patients with DRE as well as in a preclinical model of epilepsy. Synaptic terminals (synaptosomes) were obtained from the epileptic neocortex of patients with drug-resistant temporal lobe epilepsy (DR-TLE, n = 10) or drug-resistant extratemporal lobe epilepsy (DR-ETLE, n = 10) submitted to epilepsy surgery. Synaptosomes highly purified by Percoll-sucrose density gradient were characterized by confocal microscopy and Western blot. Synaptosomes were used to estimate the high KCl (33 mM)-evoked glutamate release in the presence of CBD at different concentrations. Our results revealed responsive tissue obtained from seven patients with DR-TLE and seven patients with DR-ETLE. Responsive tissue showed lower glutamate release (p < 0.05) when incubated with CBD at low concentrations (less than 100 µM) but not at higher concentrations. Tissue that was non-responsive to CBD (DR-TLE, n = 3 and DR-ELTE, n = 3) showed high glutamate release despite CBD exposure at different concentrations. Simultaneously, a block of the human epileptic neocortex was used to determine its viability through whole-cell and extracellular electrophysiological recordings. The electrophysiological evaluations supported that the responsive and non-responsive human epileptic neocortices used in the present study exhibited proper neuronal viability and stability to acquire electrophysiological responses. We also investigated whether the subchronic administration of CBD could reduce glutamate over-release in a preclinical model of temporal lobe epilepsy. Administration of CBD (200 mg/kg, p.o. every 24 h for 7 days) to rats with lithium-pilocarpine-evoked spontaneous recurrent seizures reduced glutamate over-release in the hippocampus. The present study revealed that acute exposure to low concentrations of CBD can reduce the glutamate over-release in synaptic terminals obtained from some patients with DRE. This effect is also evident when applied subchronically in rats with spontaneous recurrent seizures. An important finding was the identification of a group of patients that were non-responsive to CBD effects. Future studies are essential to identify biomarkers of responsiveness to CBD to control DRE.

Hydrogen sulfide prevents the vascular dysfunction induced by severe traumatic brain injury in rats by reducing reactive oxygen species and modulating eNOS and H2S-synthesizing enzyme expression.

AIM: To assess the effects of subchronic administration with NaHS, an exogenous H2S donor, on TBI-induced hypertension and vascular impairments. MAIN METHODS: Animals underweministration does not prevent the body weight loss but slightly imnt a lateral fluid percussion injury, and the hemodynamic variables were measured in vivo by plethysmograph method. The vascular function in vitro, the ROS levels by the DCFH-DA method and the expression of H2S-synthesizing enzymes and eNOS by Western blot were measured in isolated thoracic aortas at day 7 post-TBI. The effect of L-NAME on NaHS-induced effects in vascular function was evaluated. Brain water content was determined 7 days after trauma induction. Body weight was recorded throughout the experimental protocol, whereas the sensorimotor function was evaluated using the neuroscore test at days -1 (basal), 2, and 7 after the TBI induction. KEY FINDINGS: TBI animals showed: 1) an increase in hemodynamic variables and ROS levels in aortas; 2) vascular dysfunction; 3) sensorimotor dysfunction; and 4) a decrease in body weight, the expression of H2S-synthesizing enzymes, and eNOS phosphorylation. Interestingly, NaHS subchronic administration (3.1 mg/kg; i.p.; every 24 h for six days) prevented the development of hypertension, vascular dysfunction, and oxidative stress. L-NAME abolished NaHS-induced effects. Furthermore, NaHS treatment restored H2S-synthesizing enzymes and eNOS phosphorylation with no effect on body weight, sensorimotor impairments, or brain water content. SIGNIFICANCE: Taken together, these results demonstrate that H2S prevents TBI-induced hypertension by restoring vascular function and modulating ROS levels, H2S-synthesizing enzymes expression, and eNOS phosphorylation.

Targeting hydrogen sulfide and nitric oxide to repair cardiovascular injury after trauma.

The systemic cardiovascular effects of major trauma, especially neurotrauma, contribute to death and permanent disability in trauma patients and treatments are needed to improve outcomes. In some trauma patients, dysfunction of the autonomic nervous system produces a state of adrenergic overstimulation, causing either a sustained elevation in catecholamines (sympathetic storm) or oscillating bursts of paroxysmal sympathetic hyperactivity. Trauma can also activate innate immune responses that release cytokines and damage-associated molecular patterns into the circulation. This combination of altered autonomic nervous system function and widespread systemic inflammation produces secondary cardiovascular injury, including hypertension, damage to cardiac tissue, vascular endothelial dysfunction, coagulopathy and multiorgan failure. The gasotransmitters nitric oxide (NO) and hydrogen sulfide (H2S) are small gaseous molecules with potent effects on vascular tone regulation. Exogenous NO (inhaled) has potential therapeutic benefit in cardio-cerebrovascular diseases, but limited data suggests potential efficacy in traumatic brain injury (TBI). H2S is a modulator of NO signaling and autonomic nervous system function that has also been used as a drug for cardio-cerebrovascular diseases. The inhaled gases NO and H2S are potential treatments to restore cardio-cerebrovascular function in the post-trauma period.

Cannabidiol Reduces Short- and Long-Term High Glutamate Release after Severe Traumatic Brain Injury and Improves Functional Recovery.

This study aimed to determine if orally administered cannabidiol (CBD) lessens the cortical over-release of glutamate induced by a severe traumatic brain injury (TBI) and facilitates functional recovery. The short-term experiment focused on identifying the optimal oral pretreatment of CBD. Male Wistar rats were pretreated with oral administration of CBD (50, 100, or 200 mg/kg) daily for 7 days. Then, extracellular glutamate concentration was estimated by cortical microdialysis before and immediately after a severe TBI. The long-term experiment focused on evaluating the effect of the optimal treatment of CBD (pre- vs. pre- and post-TBI) 30 days after trauma. Sensorimotor function, body weight, and mortality rate were evaluated. In the short term, TBI induced a high release of glutamate (738% ± 173%; p < 0.001 vs. basal). Oral pretreatment with CBD at all doses tested reduced glutamate concentration but with higher potency at when animals received 100 mg/kg (222 ± 33%, p < 0.01 vs. TBI), an effect associated with a lower mortality rate (22%, p < 0.001 vs. TBI). In the long-term experiment, the TBI group showed a high glutamate concentration (149% p < 0.01 vs. SHAM). In contrast, animals receiving the optimal treatment of CBD (pre- and pre/post-TBI) showed glutamate concentrations like the SHAM group (p > 0.05). This effect was associated with high sensorimotor function improvement. CBD pretreatment, but not pre-/post-treatment, induced a higher body weight gain (39% ± 2.7%, p < 0.01 vs. TBI) and lower mortality rate (22%, p < 0.01 vs. TBI). These results support that orally administered CBD reduces short- and long-term TBI-induced excitotoxicity and facilitated functional recovery. Indeed, pretreatment with CBD was sufficient to lessen the adverse sequelae of TBI.

Exogenous hydrogen sulfide restores CSE and CBS but no 3-MST protein expression in the hypothalamus and brainstem after severe traumatic brain injury.

Hydrogen sulfide (H2S) is a gasotransmitter endogenously synthesized by cystathionine-γ-lyase (CSE), cystathionine-ß-synthase (CBS), and 3-mercaptopiruvate sulfurtransferase (3-MST) enzymes. H2S exogenous administration prevents the development of hemodynamic impairments after traumatic brain injury (TBI). Since the hypothalamus and the brainstem highly regulate the cardiovascular system, this study aimed to evaluate the effect of NaHS subchronic treatment on the changes of H2S-sythesizing enzymes in those brain areas after TBI and in physiological conditions. For that purpose, animals were submitted to a lateral fluid percussion injury, and the changes in CBS, CSE, and 3-MST protein expression were measured by western blot at days 1, 2, 3, 7, and 28 in the vehicle group, and 7 and 28 days after NaHS treatment. After severe TBI induction, we found a decrease in CBS and CSE protein expression in the hypothalamus and brainstem; meanwhile, 3-MST protein expression diminished only in the hypothalamus compared to the Sham group. Remarkably, i.p. daily injections of NaHS, an H2S donor, (3.1 mg/kg) during seven days: (1) restored CBS and CSE but no 3-MST protein expression in the hypothalamus at day 28 post-TBI; (2) reestablished only CSE in brainstem 7 and 28 days after TBI; and (3) did not modify H2S-sythesizing enzymes protein expression in uninjured animals. Mainly, our results show that the NaHS effect on CBS and CSE protein expression is observed in a time- and tissue-dependent manner with no effect on 3-MST expression, which may suggest a potential role of H2S synthesis in hypothalamus and brainstem impairments observed after TBI.

Hydrogen Sulfide Subchronic Treatment Improves Hypertension Induced by Traumatic Brain Injury in Rats through Vasopressor Sympathetic Outflow Inhibition.

Traumatic brain injury (TBI) represents a critical public health problem around the world. To date, there are no accurate therapeutic approaches for the management of cardiovascular impairments induce by TBI. In this regard, hydrogen sulfide (H2S), a novel gasotransmitter, has been proposed as a neuro- and cardioprotective molecule. This study was designed to determine the effect of subchronic management with sodium hydrosulfide (NaHS) on hemodynamic, vasopressor sympathetic outflow and sensorimotor alterations produced by TBI. Animals underwent a lateral fluid percussion injury, and changes in hemodynamic variables were measured by pletismographic methods. In addition, vasopressor sympathetic outflow was assessed by a pithed rat model. Last, sensorimotor impairments were evaluated by neuroscore test and beam-walking test. At seven, 14, 21, and 28 days after moderate-severe TBI, the animals showed: (1) a decrease on sensorimotor function in the neuroscore test and beam-walking test; (2) an increase in heart rate, systolic, diastolic, and mean blood pressure; (3) progressive sympathetic hyperactivity; and (4) a decrease in vasopressor responses induced by noradrenaline (α1/2-adrenoceptors agonist) and UK 14,304 (selective α2-adrenoceptor agonist). Interestingly, intraperitoneal daily injections of NaHS, an H2S donor (3.1 and 5.6 mg/kg), during seven days after TBI prevented the development of the impairments in hemodynamic variables, which were similar to those obtained in sham animals. Moreover, NaHS treatment prevented the sympathetic hyperactivity and decreased noradrenaline-induced vasopressor responses. No effects on sensorimotor dysfunction were observed, however. Taken together, our results suggest that H2S ameliorates the hemodynamic and sympathetic system impairments observed after TBI.

Propylparaben Reduces the Long-Term Consequences in Hippocampus Induced by Traumatic Brain Injury in Rats: Its Implications as Therapeutic Strategy to Prevent Neurodegenerative Diseases.

BACKGROUND: Severe traumatic brain injury (TBI), an important risk factor for Alzheimer's disease, induces long-term hippocampal damage and hyperexcitability. On the other hand, studies support that propylparaben (PPB) induces hippocampal neuroprotection in neurodegenerative diseases. OBJECTIVE: Experiments were designed to evaluate the effects of subchronic treatment with PPB on TBI-induced changes in the hippocampus of rats. METHODS: Severe TBI was induced using the lateral fluid percussion model. Subsequently, rats received subchronic administration with PPB (178 mg/kg, TBI+PPB) or vehicle (TBI+PEG) daily for 5 days. The following changes were examined during the experimental procedure: sensorimotor dysfunction, changes in hippocampal excitability, as well as neuronal damage and volume. RESULTS: TBI+PEG group showed sensorimotor dysfunction (p < 0.001), hyperexcitability (64.2%, p < 0.001), and low neuronal preservation ipsi- and contralateral to the trauma. Magnetic resonance imaging (MRI) analysis revealed lower volume (17.2%; p < 0.01) and great damage to the ipsilateral hippocampus. TBI+PPB group showed sensorimotor dysfunction that was partially reversed 30 days after trauma. This group showed hippocampal excitability and neuronal preservation similar to the control group. However, MRI analysis revealed lower hippocampal volume (p < 0.05) when compared with the control group. CONCLUSION: The present study confirms that post-TBI subchronic administration with PPB reduces the long-term consequences of trauma in the hippocampus. Implications of PPB as a neuroprotective strategy to prevent the development of Alzheimer's disease as consequence of TBI are discussed.

Sodium Cromoglycate Decreases Sensorimotor Impairment and Hippocampal Alterations Induced by Severe Traumatic Brain Injury in Rats.

Severe traumatic brain injury (TBI) results in significant functional disturbances in the hippocampus. Studies support that sodium cromoglycate (CG) induces neuroprotective effects. This study focused on investigating the effects of post-TBI subchronic administration of CG on hippocampal hyperexcitability and damage as well as on sensorimotor impairment in rats. In contrast to the control group (Sham+SS group), animals undergoing severe TBI (TBI+SS group) showed sensorimotor dysfunction over the experimental post-TBI period (day 2, 55%, p < 0.001; day 23, 39.5%, p < 0.001; day 30, 38.6%, p < 0.01). On day 30 post-TBI, TBI+SS group showed neuronal hyperexcitability (63.3%, p < 0.01). The hippocampus ipsilateral to the injury showed volume reduction (14.4%, p < 0.001) with a volume of damage of 0.15 ± 0.09 mm3. These changes were associated with neuronal loss in the dentate gyrus (ipsilateral, 33%, p < 0.05); hilus (ipsilateral, 77%, p < 0.001; contralateral, 51%, p < 0.001); Cornu Ammonis (CA)1 (ipsilateral, 40%, p < 0.01), and CA3 (ipsilateral, 52%, p < 0.001; contralateral, 34%, p < 0.01). Animals receiving subchronic treatment with CG (50 mg/kg, s.c. daily for 10 days) after TBI (TBI+CG group) displayed a sensorimotor dysfunction less evident than that of the TBI+SS group (p < 0.001). Their hippocampal excitability was similar to that of the Sham+SS group (p = 0.21). The TBI+CG group presented hippocampal volume reduction (12.7%, p = 0.94) and damage (0.10 ± 0.03 mm3, p > 0.99) similar to the TBI+SS group. However, their hippocampal neuronal preservation was similar to that of the Sham+SS group. These results indicate that CG represents an appropriate and novel pharmacological strategy to reduce the long-term sensorimotor impairment and hippocampal damage and hyperexcitability that result as consequences of severe TBI.