Rat Brainstem Research Articles

Simultaneous Determination of Substance P and CGRP in Rat Brainstem Tissue Using LC-ESI-MS/MS Method

Background: Substance P and CGRP are neuropeptides that belong to the thachykinin and calcitonin family, respectively. They play an important role in neuropathic pain development and regulation. Methods: In this study, simultaneous analysis of Substance P and CGRP in rat brainstem tissue was conducted by using LC-ESI-MS/MS method. A gradient mobile phase was used with a Zorbax C8 150x2.1 mm, a particle size of 3.5 µm narrow-bore column. Mobile phases consist of 0.2% formic acid in water (A) and 0.2% formic acid in acetonitrile (v/v) (B). Firstly, mass spectrometry parameters were optimized. After achieving chromatographic separation, the method has been validated and applied to rat brainstem samples. Results: SP and CGRP were eluted at 8.7 min and 10.1 min, respectively. Mass spectrum of neuropeptides was obtained in positive polarity and ion transitions of Substance P and CGRP were monitored at m/z 674.4→[600+254.4] and 952.2→1214.200, respectively. Linearity range for quantification was 5.85-438.52 ng/mL for Substance P and 0.21-155.28 ng/mL for CGRP. The limit of quantification was 5.85 ng/mL for Substance P and 0.21 ng/mL for CGRP. According to tissue samples analysis (n=6), basal endogen concentrations of Substance P and CGRP in brainstem tissue were calculated as 260.57±11.24 ng/g and 22.53±0.75 ng/g respectively. Conclusion: A novel LC-ESI-MS/MS method was successfully developed and validated according to the bioanalytical validation guideline published by the EMA including precision, accuracy, specificity and linearity. Acceptable results were achieved. Basal endogen concentrations of Substance P and CGRP in brainstem tissue were determined. Keywords: Substance P, CGRP, LC-ESI-MS/MS, rat brainstem tissue, neuropeptide, determination.

Purinergic modulation of neuronal activity in the rat prepositus hypoglossi nucleus.

In the nervous system, adenosine 5'-trisphosphate (ATP) functions as a neurotransmitter and binds to ionotropic P2X receptors and metabotropic P2Y receptors. Although ATP receptors are expressed in the prepositus hypoglossi nucleus (PHN), which is a brainstem structure involved in controlling horizontal gaze, it is unclear whether ATP indeed affects the activity of PHN neurons. In this study, we investigated the effects of ATP on spontaneous firing of PHN neurons using whole-cell recordings in rat brainstem slices. Bath application of ATP increased or decreased the spontaneous firing rate of the neurons in a dose-dependent manner, indicating that ATP indeed affects PHN neuronal activity. To clarify the mechanisms of the ATP effects, we investigated the current responses of PHN neurons to a local application of ATP. The ATP application induced a fast inward (FI) current, a slow inward (SI) current, and/or a slow outward (SO) current in the neurons. The agonists of P2X and P2Y receptors induced FI and SI currents, respectively. The SO currents were not induced by the ATP agonists but were induced by adenosine, which may be extracellularly converted from ATP by ectonucleotidases. An antagonist of adenosine P1 (A1 ) receptors abolished the adenosine-induced SO currents and bath application of adenosine decreased the spontaneous firing rate of all PHN neurons tested. These results indicate that PHN neurons express functional purinoceptors and show that the FI, SI, and SO currents were mediated via P2X, P2Y, and A1 receptors, respectively.

Selective modulator of peroxisome proliferator-activated receptor-α protects propionic acid induced autism-like phenotypes in rats

AimsThe present study investigated the neuropharmacological role of PPAR-α modulator, fenofibrate in postnatal-propionic acid induced symptomatology related with autism spectrum disorders (ASD) in Wistar rats. Main methodsThe propionic acid (250 mg/kg, p.o.) was administered to rats from postnatal 21st day to 23rd day to induce autism-related neurobehavioral and neurobiochemical alterations in rats. Then, rats were treated with fenofibrate (100 mg/kg and 200 mg/kg, orally) from postnatal 24th day till 48th day. The social behavior (three chambers social testing apparatus), repetitive behavior (Y-maze), locomotor activity (actophotometer), anxiety (elevated plus maze) and exploratory behavior (hole board test) were assessed. Biochemically, oxidative stress (thiobarbituric acid reactive species and reduced glutathione level) and neuroinflammation (interleukin-6, tumor necrosis factor-α and interleukin-10) were evaluated in the cerebellum, brainstem and prefrontal cortex of rats. Key findingsPropionic acid-treated rats showed social impairment, repetitive behavior, hyperlocomotion, anxiety and low exploratory activity. Also, these animals showed higher levels of oxidative stress (increased in thiobarbituric acid reactive species and decreased in reduced glutathione level) as well as inflammation (increased in interleukin-6, tumor necrosis factor-α and decreased in interleukin-10) and inflammation in aforementioned brain-regions. Treatment with fenofibrate significantly attenuated the propionic acid induced-social impairment, repetitive behavior, hyperactivity, anxiety and low exploratory activity. Furthermore, fenofibrate also reduced the oxidative stress and neuroinflammation in propionic acid-treated rats. SignificanceA selective PPAR-α agonist, fenofibrate provides neurobehavioral and neurobiochemical benefits in postnatal-propionic acid induced autism-related phenotype in rats. Thus, fenofibrate may further be studied for its possible benefits in ASD symptoms.

Kinetic properties of persistent Na+ current orchestrate oscillatory bursting in respiratory neurons.

The rhythmic pattern of breathing depends on the pre-Bötzinger complex (preBötC) in the brainstem, a vital circuit that contains a population of neurons with intrinsic oscillatory bursting behavior. Here, we investigate the specific kinetic properties that enable voltage-gated sodium channels to establish oscillatory bursting in preBötC inspiratory neurons, which exhibit an unusually large persistent Na+ current (INaP). We first characterize the kinetics of INaP in neonatal rat brainstem slices in vitro, using whole-cell patch-clamp and computational modeling, and then test the contribution of INaP to rhythmic bursting in live neurons, using the dynamic clamp technique. We provide evidence that subthreshold activation, persistence at suprathreshold potentials, slow inactivation, and slow recovery from inactivation are kinetic features of INaP that regulate all aspects of intrinsic rhythmic bursting in preBötC neurons. The slow and cumulative inactivation of INaP during the burst active phase controls burst duration and termination, while the slow recovery from inactivation controls the duration of the interburst interval. To demonstrate this mechanism, we develop a Markov state model of INaP that explains a comprehensive set of voltage clamp data. By adding or subtracting a computer-generated INaP from a live neuron via dynamic clamp, we are able to convert nonbursters into intrinsic bursters, and vice versa. As a control, we test a model with inactivation features removed. Adding noninactivating INaP into nonbursters results in a pattern of random transitions between sustained firing and quiescence. The relative amplitude of INaP is the key factor that separates intrinsic bursters from nonbursters and can change the fraction of intrinsic bursters in the preBötC. INaP could thus be an important target for regulating network rhythmogenic properties.

Optical analysis of functional development of the facial motor nucleus in the embryonic rat brainstem.

Facial motor neurons of the rat embryo are first generated in rhombomere 4 and then migrate in the caudo-ventral direction. This migration forms a unique axonal trajectory called the genu, a loop of facial motor axons around the abducens nucleus. It is still unclear when and how this unique structure is functionally established during ontogenesis. Using voltage-sensitive dye (VSD) recording and the DiI staining method, we identified neural responses evoked by facial nerve (N.VII) stimulation and examined developmental processes of the facial motor nucleus in E12-E17 rat brainstems. We identified two types of fast spike-like signals; a long-duration signal, which corresponded to the action potential in the N.VII soma, and a short-duration signal, which reflected the action potential in the N.VII axons. The long-duration signal was detected as early as E13, suggesting that the N.VII motor neuron is already excitable at the beginning of cell migration. The response area of the long-duration signal extended caudally at E13-E14, and shifted in a ventral direction at E15. At E16-E17, the long-duration signal was concentrated in the caudo-ventral area, which was comparable to the location of the facial motor nucleus in the adult rat brainstem. These results demonstrate that developmental processes of cell migration and nuclear organization can be visualized and identified functionally with the VSD recording. We discuss the results by comparing functiogenesis and morphogenesis of the N.VII pathway.

Serotonin transporter inhibition during neonatal period induces sex-dependent effects on mitochondrial bioenergetics in the rat brainstem.

The serotonin reuptake is mainly regulated by the serotonin transporters (SERTs), which are abundantly found in the raphe nuclei, located in the brainstem. Previous studies have shown that dysfunction in the SERT has been associated with several disorders, including depression and cardiovascular diseases. In this manuscript, we aimed to investigate how gender and the treatment with a serotonin selective reuptake inhibitor (SSRI) could affect mitochondrial bioenergetics and oxidative stress in the brainstem of male and female rats. Fluoxetine, our chosen SSRI, was used during the neonatal period (i.e., from postnatal Day 1 to postnatal Day 21-PND1 to PND21) in both male and female animals. Thereafter, experiments were conducted in adult rats (60days old). Our results demonstrate that, during lactation, fluoxetine treatment modulates the mitochondrial bioenergetics in a sex-dependent manner, such as improving male mitochondrial function and female antioxidant capacity.

Rat Brainstem Hemorrhage Model: Key Points to Success in Modeling

A successful animal brainstem hemorrhage model can be used to study pathophysiology and molecular mechanisms of neurologic deterioration or recovery and therapeutic interventions after the brainstem hemorrhage. Herein, we provide a reproducible and reliable model for rat brainstem hemorrhage and summarize our key points to success in modeling. Twenty-four adult male Sprague-Dawley rats were divided into 3 groups and were injected with 30, 40, and 60 μL autologous blood into the right pontine basal tegmentum after being sedated. The initial model was evaluated with neurobehavior testing, autopsy, and histopathology. The modeling conditions were modified in Group IV rats, and 20 more rats were used to check the reproducibility and reliability of the modified model. Finally, magnetic resonance imaging (MRI) was used to confirm the brainstem hemorrhage. Only 6 rats had significant pontine hemorrhage in initial 3 groups on autopsy. Whereas after the modification, all Group IV rats had serious pontine hemorrhage, and modified conditions model had a 90% success rate. MRI confirmed a stable hematoma in the right basal tegmental pons with oval shape 24 hours after of blood injection. Histopathologically, there was significant perihematomal swelling, migration of inflammatory cells and structural changes in neuronal shape and contents. An ideal and repeatable rat brainstem hemorrhage model by injecting autologous blood stereotactically was created, and it mimics the natural onset of brainstem hemorrhage. This brainstem hemorrhage model shows promise that providing a better way for neurology researchers to investigate pathophysiologic procedure of brainstem intracranial hemorrhage and further explore the treatment.

Synthesis of Deuterium- or Tritium-Labeled Norepinephrine and Evaluation of Its Biological Activity

A labeled norepinephrine analogue was prepared by bromination of norepinephrine with subsequent debromination with tritium or deuterium gas. The yield of tritiated norepinephrine was 10–20% with a molar radioactivity of 27 Ci/mmol. The biological activity of tritiated norepinephrine was evaluated by analyzing its specific capture by noradrenergic neutrons in thick sections of the brainstem and midbrain of Wistar rats, and in a suspension of cells of pheochromocytoma PC12 expressing the membrane transporter of norepinephrine. It is shown that tritium-labeled norepinephrine synthesized by the proposed method does not differ in biological activity from the natural norepinephrine.

TRK-fused Gene Protein Localization Is Prominent in Serotonergic and Noradrenergic Cell Groups, and Some Lower Motor Neurons in the Corticospinal Tract of the Rat Brainstem.

The TRK-fused gene (TFG) is reported to be involved in the regulation of cell size, apoptosis, cell growth, ER-Golgi protein secretion, NF-κβ pathway signaling, the ubiquitin-proteasome system, and pancreatic β-cell mass and function. TFG mutations were reported in some neurodegenerative diseases affecting sensory and motor functions. However, the function of TFG in the nervous system and how TFG mutations lead to neurodegeneration remain unclear. In this study, we employed double immunohistochemistry to investigate the details of TFG localization patterns in monoaminergic and cholinergic neurons in the brainstem. Intense TFG immunoreactivity was observed in the dorsal raphe nucleus, the locus coeruleus, and the ventral horn of the spinal cord. TFG immunoreactivity was observed in some serotonergic neurons in all B1–B9 cell groups, and some noradrenergic neurons in all A1–A7 cell groups in the rat brainstem, while no immunoreactivity was observed in the dopaminergic neurons in A8–A10 cell groups. TFG immunoreactivity was observed in all ChAT-positive motor nuclei in the lower corticospinal tract of the rat brainstem.

Defining mechanisms of neural plasticity after brainstem ischemia in rats.

Neurological recovery after stroke mainly depends on the location of the lesion. A substantial portion of strokes affects the brainstem. However, patterns of neural plasticity following brainstem ischemia are almost unknown. Here, we established a rat brainstem ischemia model that resembles key features of the human disease and investigated mechanisms of neural plasticity, including neurogenesis and axonal sprouting as well as secondary neurodegeneration. Spontaneous functional recovery was accompanied by a distinct pattern of axonal sprouting, for example, an increased bilateral fiber outgrowth from the corticorubral tract to the respective contralesional red nucleus suggesting a compensatory role of extrapyramidal pathways after damage to pyramid tracts within the brainstem. Using different markers for DNA replication, we showed that the brainstem displays a remarkable ability to undergo specific plastic cellular changes after injury, highlighting a yet unknown pattern of neurogenesis. Neural progenitor cells proliferated within the dorsal brainstem and migrated toward the lesion, whereas neurogenesis in classic neurogenic niches, the subventricular zone of the lateral ventricle and the hippocampus, remained, in contrast to what is known from hemispheric stroke, unaffected. These beneficial changes were paralleled by long-term degenerative processes, that is, corticospinal fiber loss superior to the lesion, degeneration of spinal tracts, and a decreased neuron density within the ipsilesional substantia nigra and the contralesional red nucleus that might have limited further functional recovery. Our findings provide knowledge of elementary plastic adaptions after brainstem stroke, which is fundamental for understanding the human disease and for the development of new treatments. Ann Neurol 2018;83:1003-1015.

Increased astrocytic marker expression in the brainstem in rats with a mutation in the inwardly rectifying potassium channel 5.1 (Kir5.1)

Inwardly rectifying potassium (Kir) channels modulate membrane potentials and contribute to K+ recycling in the kidney and brain. We recently showed in rats with mutations in Kcnj16 (SSKcnj16−/− rats), the gene that encodes Kir5.1 channels, lead to dysfunction in renal salt handling, prevents salt‐sensitive hypertension, but also leads to sudden unexpected death during salt‐loading (Palygin et al., JCI Insight 2017). In addition, SSKcnj16−/− rats show renal and ventilatory pH regulation dysfunction, and have audiogenic seizures. Given that Kir4.1 and Kir5.1 channels are thought to be co‐expressed in CNS astrocytes and drive K+ recycling in the CNS, we sought to test the hypothesis that SSKcnj16−/− rats will have an astrocytic expansion within brainstem nuclei that contribute to ventilatory control mechanisms. To test this hypothesis, we used an immunohistochemical approach to assess neuronal cell counts (NeuN) and the density of expression of an astrocytic marker (GFAP) within multiple nuclei in the brainstem of adult (>8 weeks of age) naïve control SS rats (n=2), in addition to SSKcnj16−/− rats with and without (naïve; n=3) audiogenic seizure stimulation once/day for 3 consecutive days. Rats were anesthetized, transcardially perfused and fixed (4% PFA), cryoprotected in sucrose before frozen sectioning (20μm thickness) and placement on coated slides. Slides were incubated sequentially with anti‐GFAP and anti‐NeuN antibodies which were detected with 594 anti‐rabbit and 488 anti‐mouse as secondary antibodies, respectively, and images obtained using standard epifluorescence microscopy and analyzed using image J within which an algorithm was developed to count NeuN positive neurons and to determine the percent of background positive for GFAP. The nuclei analyzed were: Raphe Pallidus (RPa), Obscurus (Rob) and Magnus (Rm), hypoglossal motor nuclei (XII), dorsal motor nucleus of the vagus (DMV), nucleus tractus solitarus (NTS), nucleus ambiguous (AMB), the ventral respiratory column (VRC) and cochlear (Co) nuclei. In control SS rats, we noted that the relative percentage of each area stained positive for GFAP was 17.2% (RPa), 7.8 (Rob), 6.4 (Rm), 5.0 (XII), 5.6 (DMV), 6.0 (NTS), 4.6 (AMB), 4.8 (VRC) and 15.6 (Co) compared to the naïve SSKcnj16−/− rats 30.1, 14.1, 16, 17, 11.5, 13.8, 13.1, 15 and 24 respectively. An unpaired t‐test analysis was performed, and statistically significant differences were found for the AMB nucleus (p=0.035). Ongoing experiments will determine if additional animals in each group will resolve other potential differences in neurons and/or astrocytes among naïve control and SSKcnj16−/− rats, or if there are additional alterations in brainstem neurons/glia after multiple audiogenic seizures, which may induce reactive gliosis. We conclude that mutations in Kir5.1 channels may alter astrocytes but not neuron counts within respiratory nuclei.Support or Funding InformationSupported by NIH HL122358This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ang II‐Mediated Regulation of AGT Protein Synthesis in Rat Brainstem Astrocytes is βarrestin1 Dependent

ObjectiveStudy the role of βarrestins in Angiotensin (Ang) II‐mediated regulation of angiotensinogen (AGT) protein synthesis in astrocytes isolated from the brainstem of spontaneously hypertensive rats (SHRs) or normotensive rats.BackgroundThe βarrestins (βarrestin1 and‐2) terminate G protein signaling by G protein‐coupled receptors (GPCRs) and, at the same time, they initiate their own “second wave” of signaling from GPCRs, independently of G proteins. Ang II, through its cognate GPCR, Angiotensin II type1 receptor (AT1R), signals via both G proteins and βarrestins in various cell types. In our previous study, we reported that SII (an Ang II peptide analog which is a βarrestin‐biased agonist), similarly to Ang II, upregulates AGT synthesis in a time‐dependent manner. Here, we sought to delineate whether Ang II‐mediated regulation of AGT protein synthesis in brainstem‐derived astrocytes is G protein or βarrestin or both G protein and βarrestin dependent.MethodsPrimary cultures of brainstem astrocytes were isolated from the brains of 2–3 days old pups. Double‐stranded siRNAs (Dharmacon) targeting rat βarrestin1 or βarrestin2 were used to knockdown βarrestins. Non‐silencing siRNA (Dharmacon) was used as a control. RNiMAX (Invitrogen) was used as a transfection reagent. Ang II (100 nM) and 10 μM SII (the βarrestin‐biased agonist) were used to study AT1R‐mediated AGT protein expression. Protein analysis was done using Western blotting and mRNA measurement was done using quantitative real‐time PCR. The results in SHR astrocytes were compared to normotensive rat astrocytes.ResultsOur results indicate that βarrestin1 is the major βarrestin isoform in astrocytes isolated from both SHR and normotensive rat brainstem astrocytes. Ang II upregulated AGT protein synthesis in both SHR and normotensive rat astrocytes. This effect was higher in SHR as compared to normotensive rats. siRNA mediated knockdown of βarrestin1 abolished Ang II‐mediated AGT protein synthesis. However, βarrestin2 knockdown did not inhibit Ang II‐mediated AGT protein synthesis.ConclusionIn this study, we showed that βarrestin1 plays a major role in the Ang II‐AT1R‐mediated regulation of AGT protein synthesis in rat brainstem astrocytes in both SHR and normotensive rat models, which could be an important therapeutic target for the treatment of hypertension and other cardiovascular diseases.Support or Funding InformationFunding for this project was provided through a Nova Southeastern University President's Faculty Research and Development Grant (PFRDG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interaction between the Dopamine D4 Receptor and the Angiotensin II Type 1 Receptor in Rat Astrocytes

ObjectiveTo determine the basal expression of the Dopamine D4 receptor (D4R) in Wistar rat astrocytes and investigate if Angiotensin (Ang) II can regulate D4R expression.BackgroundThe interaction between the renin angiotensin system (RAS) and the dopaminergic system in the regulation of peripheral blood pressure is well established; however, such interaction in the brain is not studied. Our goal is to investigate whether there is an interaction between these two systems in a rat model brain system.MethodsBrainstem and cerebellum astrocytes were prepared from 2–3 days Wistar rat pups. These brain regions were selected because they express RAS components. Quiescent cells were treated with 100nM Ang II for 1 to 48 hours. We determined D4R and AT1R, protein levels by western blots and mRNA levels by quantitative real time polymerase chain reaction.ResultsWistar cerebellum astrocytes had significantly higher basal D4R protein levels compared to Wistar brainstem. Basal D4R mRNA levels in cerebellar astrocytes were higher than the basal D4R mRNA levels in brainstem astrocytes. Basal AT1R protein expression was significantly higher in brainstem astrocytes compared to cerebellar astrocytes. Ang II reduced the D4R protein levels relative to untreated controls in Wistar brainstem at most of the time points examined. There were no significant differences in D4R protein levels in cerebellum astrocytes, relative to controls after Ang II treatment.ConclusionWe demonstrated the ability of Ang II to reduce the D4R protein levels in Wistar rat brainstem samples. Our results showed that under physiological conditions, D4Rs in brainstem astrocytes were more sensitive to Ang II treatments compared to those in cerebellum. This could be attributed to the role of the brainstem in blood pressure regulation. Indeed, brainstem astrocytes had higher AT1R protein levels compared to cerebellum astrocytes. This interaction between the AT1R and the D4R is a potential therapeutic target for pathophysiological conditions mediated by these two systems. Further studies will investigate this interaction in astrocytes and neurons isolated from spontaneously hypertensive rats, a rat model of Hypertension and Attention Deficit Hyperactivity Disorder.Support or Funding InformationThis study was funded through a President's Faculty Research and Development Grant from Nova Southeastern University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Suction electrode recording in locus coeruleus of newborn rat brain slices reveals network bursting comprising summated non-synchronous spiking

The brainstem locus coeruleus (LC) controling behaviors like arousal, sleep, breathing, pain or opioid withdrawal is an established model for spontaneous action potential synchronization. Such synchronous ‘spiking’ might produce an extracellular field potential (FP) which is a crucial tool for neural network analyses. We found using ≥10 μm tip diameter suction electrodes in newborn rat brainstem slices that the LC generates at ∼1 Hz a robust rhythmic FP (rFP). During distinct rFP phases, LC neurons discharge with a jitter of ±33 ms single spikes that summate to a ∼200 ms-lasting population burst. The rFP is abolished by blocking voltage-gated Na+ channels with tetrodotoxin (TTX, 50 nM) or gap junctions with mefloquine (100 μM) and activating μ-opioid receptors with [D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin (DAMGO, 1 μM). Raising superfusate K+ from 3 to 7 mM either increases rFP rate or transforms its pattern to slower and longer multipeak bursts similar to those during early recovery from DAMGO. The results show that electrical coupling of neonatal LC neurons does not synchronize their spiking as previously proposed. They also indicate that both increased excitability (by elevated K+) and recovery from inhibition (by opioids) can enhance spike desynchronization to transform the population burst pattern. Both observations show that this gap junction-coupled neural network has a more complex connectivity than currently assumed. These new findings along with the inhibitory drug effects that are in line with previous reports based on single neuron recording point out that field potential analysis is pivotal to further the understanding of this brain circuit.

Expression of the transcription factor FOXP2 in brainstem respiratory circuits of adult rat is restricted to upper-airway pre-motor areas

Expression of the transcription factor FOXP2 is linked to brain circuits that control motor function and speech. Investigation of FOXP2 protein expression in respiratory areas of the ponto-medullary brainstem of adult rat revealed distinct rostro-caudal expression gradients. A high density of FOXP2 immunoreactive nuclei was observed within the rostral pontine Kölliker-Fuse nucleus, compared to low densities in caudal pontine and rostral medullary respiratory nuclei, including the: (i) noradrenergic A5 and parafacial respiratory groups; (ii) Bötzinger and pre-Bötzinger complex and; (iii) rostral ventral respiratory group. Moderate densities of FOXP2 immunoreactive nuclei were observed in the caudal ventral respiratory group and the nucleus retroambiguus, with significant density levels found in the caudal half of the dorsal respiratory group and the hypoglossal pre-motor area lateral around calamus scriptorius. FOXP2 immunoreactivity was absent in all cranial nerve motor nuclei. We conclude that FOXP2 expression in respiratory brainstem areas selectively delineates laryngeal and hypoglossal pre-motor neuron populations essential for the generation of sound and voice.

Tualang Honey Reduced Neuroinflammation and Caspase-3 Activity in Rat Brain after Kainic Acid-Induced Status Epilepticus.

The protective effect of tualang honey (TH) on neuroinflammation and caspase-3 activity in rat cerebral cortex, cerebellum, and brainstem after kainic acid- (KA-) induced status epilepticus was investigated. Male Sprague-Dawley rats were pretreated orally with TH (1.0 g/kg body weight) five times at 12 h intervals. KA (15 mg/kg body weight) was injected subcutaneously 30 min after last oral treatment. Rats were sacrificed at 2 h, 24 h, and 48 h after KA administration. Neuroinflammation markers and caspase-3 activity were analyzed in different brain regions 2 h, 24 h, and 48 h after KA administration. Administration of KA induced epileptic seizures. KA caused significant (p < 0.05) increase in the level of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), glial fibrillary acidic protein (GFAP), allograft inflammatory factor 1 (AIF-1), and cyclooxygenase-2 (COX-2) and increase in the caspase-3 activity in the rat cerebral cortex, cerebellum, and brainstem at multiple time points. Pretreatment with TH significantly (p < 0.05) reduced the elevation of TNF-α, IL-1β, GFAP, AIF-1, and COX-2 level in those brain regions at multiple time points and attenuated the increased caspase-3 activity in the cerebral cortex. In conclusion, TH reduced neuroinflammation and caspase-3 activity after kainic acid- (KA-) induced status epilepticus.

Gliotoxic Injury in the Rat Brainstem is Ameliorated by Curcumin

Gliotoxic Injury in the Rat Brainstem is Ameliorated by Curcumin

Expression of genes for neurotransmitter transporters in astrocytes in different brain regions in experiment

To investigate the expression of transporters of different neurotransmitters (glutamate, aspartate, lactate, choline) in the culture of astrocytes isolated from different regions of the brain (cortex, hippocampus and brainstem) in 3- and 11-day rats. An experimental study was performed on 24 3- (n=12) and 11-days (n=12) old rats (Rattus norvegicus). The results of high-performance sequencing were analyzed. The expression of glutamate and aspartate transporters in the brainstem of 3-day rats was higher than in other regions, however, an opposite effect was observed in 11-day rats. The expression of lactate transporters with age became identical to those of the cortex. The data demonstrate the particular qualities of neuro-astrocytic connections and the important role of astrocytes in signal transmission. Results of the study performed by using genetic methods developed by the authors for the study of neurotransmitter transporters make it possible to recommend these methods to control the neurogenesis and neurohomeostasis, including in cerebrovascular and neurodegenerative diseases.

Oxidative stress and Na,K-ATPase activity differential regulation in brainstem and forebrain of Wistar Audiogenic rats may lead to increased seizure susceptibility

The Wistar Audiogenic Rat (WAR) is a well-characterized seizure-prone, inbred rodent strain that, when acutely stimulated with high-intensity sounds, develops brainstem-dependent tonic-clonic seizures that can evolve to limbic-like, myoclonic (forebrain) seizures when the acoustic stimuli are presented chronically (audiogenic kindling). In order to investigate possible mechanisms underlying WAR susceptibility to seizures, we evaluated Na,K-ATPase activity, Ca-ATPase activity, Mg-ATPase activity, lipid membrane composition and oxidative stress markers in whole forebrain and whole brainstem samples of naïve WAR, as compared to samples from control Wistar rats. We also evaluated the expression levels of α1 and α3 isoforms of Na,K-ATPase in forebrain samples. We observed increased Na,K-ATPase activity in forebrain samples and increased oxidative stress markers (lipid peroxidation, glutathione peroxidase and superoxide dismutase) in brainstem samples of WAR. The Ca-ATPase activity, Mg-ATPase activity, lipid membrane composition and expression levels of α1 and α3 isoforms of Na,K-ATPase were unaltered. In view of previous data showing that the membrane potentials from naïve WAR’s neurons are less negative than that from neurons from Wistar rats, we suggest that Na,K-ATPase increased activity might be involved in a compensatory mechanism necessary to maintain WAR’s brains normal activity. Additionally, ongoing oxidative stress in the brainstem could bring Na,K-ATPase activity back to normal levels, which may explain why WAR’s present increased susceptibility to seizures triggered by high-intensity sound stimulation.

TUALANG HONEY ATTENUATES KAINIC ACID-INDUCED OXIDATIVE STRESS IN RAT CEREBELLUM AND BRAINSTEM

Objective: The present study examined the protective effect of tualang honey (TH) against kainic acid (KA)-induced oxidative stress in the cerebellum and brainstem of rats.Methods: Male Sprague-Dawley rats were randomly divided into four groups: Control, KA-treated, TH+KA-treated, and topiramate (TPM, an antiepileptic agent)+KA-treated groups. Rats were pretreated orally with drinking water, TH (1.0 g/kg body weight), or TPM (40 mg/kg body weight), respectively, five times at 12 h intervals. Saline or KA (15 mg/kg body weight) were injected subcutaneously 30 min after last oral treatment. Rats were sacrificed at 2 h, 24 h, and 48 h after KA administration. Oxidative stress markers were analyzed in different brain regions (cerebellum and brainstem) 2 h, 24 h, and 48 h after KA administration.Results: KA caused significant (p&lt;0.05) elevation in the thiobarbituric acid reactive substances level, protein carbonyl contents, and nitric oxide production, impairment of glutathione system, and a significant reduction in the total antioxidant status in the rat cerebellum and brainstem at multiple time-points, as compared to control groups. Pretreatment with TH significantly (p&lt;0.05) reduced the elevation in the thiobarbituric acid reactive substances level, protein carbonyl contents, and nitric oxide production and increasing a reduction in the total antioxidant status in the rat cerebellum and brainstem induced by KA at multiple time-points, as compared to KA only-treated group.Conclusion: Taken together, this study suggests that TH has therapeutic potential in reducing oxidative stress in the cerebellum and brainstem of KA-induced rats via its antioxidant property.