Rat Brainstem Research Articles

Effects of serotonergic manipulation in the brainstem and hypothalamus of overnourished rats during lactation

Previous studies suggest that being overweight and obese is capable of altering brain function during critical periods due to the higher plasticity of the brain during development. However, more literature still needs to be on the immediate effect of overnutrition and serotonin modulation in brain areas. In this study, we aimed to evaluate the effects of serotoninergic manipulation on oxidative stress and pro-inflammatory markers in the brainstem and hypothalamus of overnourished rats. The fluoxetine treatment was performed from postnatal day 3 (PND3) to postnatal day 21 (PND21) to evaluate mitochondrial function, oxidative balance, and the mRNA levels of monoaminergic molecules such as serotonin and dopamine, pro-inflammatory cytokines, and BDNF. Neonatal overnutrition induces molecular and biochemical changes in the brainstem and hypothalamus. These nutritional disturbances during lactation dysregulate energy balance and cellular redox, inducing oxidative stress. Conversely, modulation of the serotoninergic system through pharmacological use of fluoxetine was able to reverse the deleterious effects of overnutrition during lactation, enabling better brain development and delaying the development of pathologies and oxidative stress.

The involvement of proinflammatory cytokines in the pathogenesis of audiogenic epilepsy

Epilepsy is a heterogeneous disease, which determines the relevance of investigating the mechanisms of pathogenesis of its various types, including reflex epilepsy. Pharmacotherapy is common for the treatment of patients with epilepsy, however, despite the significant recent achievements, 20-30% of patients remain resistant to the ongoing treatment. The urgency of creating new antiepileptic drugs, in particular, immune ones is due not only to a significant proportion of pharmacoresistant cases, but also to the struggle for the quality of life of patients. The neuroinflammation system in animals with different genetically determined audiogenic epilepsy proneness was investigated. These genetic groups were Krushinsky–Molodkina rats (tonic seizures of maximum intensity in response to the action of sound) and “0” strain (control group, non-convulsive phenotype). The main proinflammatory cytokines levels in the dorsal striatum and brain stem in rats of these genetic groups were measured by multiplex immunofluorescence assay. Background levels of IL-1â, IL-6 and TNFá in the dorsal striatum of KM rats were significantly lower than in the control “0” strain rats, whereas in the brain stem in the “background” levels of these metabolites did not differ. Four hours after the sound exposure, the TNFá level in the dorsal striatum of KM rats was significantly lower than in “0” rats. In KM rats, after the sound exposure and subsequent tonic seizures, the levels of IL-1â and IL-6 in the dorsal striatum were significantly higher than in the background. The IL-2 content was not detected in the background in KM rats, whereas after audiogenic seizures its level was 14.01 pg/ml. In the brain stem of KM rats, the levels of IL-1â and TNFá after audiogenic seizures were significantly lower than in the background. In rats of the “0” strain, cytokine levels in the dorsal striatum after the sound exposure did not differ from those in the background, while IL-1ß levels in their brain stem were significantly lower than the background state. Particular modulating role of the studied proinflammatory cytokines in the pathogenesis of audiogenic epilepsy is assumed, as well as certain possibility of anti-inflammatory and immune drugs application in anticonvulsant and antiepileptic therapy.

Long‐lasting behavioral and neurochemical effects of early‐life environmental enrichment in rats submitted to neonatal morphine administration

AbstractThe present study examined the medium‐ and long‐term effects of early environmental enrichment (EE) on neuromotor, nociceptive, cognitive, behavioral, and neurochemical parameters in newborn rats repeatedly exposed to morphine. The study employed 90 Wistar rats: 10 adult nulliparous females and 80 male pups. Litter was split into standard and EE housing. Following, half of each litter received saline (S) or morphine (M) injections, resulting in four groups: SC + S, EE + S, SC + M, and EE + M. EE was applied from PND1 to PND21, while morphine or saline was given daily (5 μg/s.c.) from PND8 to PND14. Neuromotor development was similar between groups. In the OF test, morphine reduced outer and total crossings, whereas EE increased inner crossings and rearings. Adult rats showed a decrease in outer and total crossings and grooming and an increase in rearing. EE increased the number of protected and unprotected head dipping. Adult rats showed an increase in protected head dipping. Adult rats showed a lower recognition index, and, when exposed to EE, a lower anxiety index and analgesia. EE increased brainstem and hippocampal BDNF levels. Adult rats had increased hypothalamus, spinal cord, and brainstem BDNF levels, an increase in the spinal cord, and decreased hypothalamus TNF‐α levels. This study demonstrated that early‐life EE raises BDNF levels in the brainstem and hippocampus of rats and modifies their behaviors (such as nociception, exploration, and anxiety) in a state‐dependent manner (morphine and age).

Brainstem transcriptomic changes in male Wistar rats after acute stress, comparing the use of duplex specific nuclease (DSN)

In this work, we have analyzed the transcriptomic changes in the brainstem of male Wistar rats 2 h after an acute stress exposure. We performed duplex-specific nuclease normalization of cDNA libraries and compared the results back-to-back for the first time. Based on our RNAseq data, we selected reference genes for RT-qPCR that are best suited for acute stress experiments. Most genes were upregulated. We detected a massive shift in neuropeptide Crh, Trh,Cga, Tshb, Uts2b, Tac4, Lep and neuropeptide receptor Hcrtr1, Sstr5, Bdkrb2, Crhr2 signaling, as well as glutamate Grin3b, Grm2 and GABA Gpr156, acetylcholine Chrm4,Chrne, adrenergic Adra2b receptors expression. A strong increase in the expression of intermediate filaments Krt83/Krt86/Krt80/Krt84/Krt87/Krt4/Krt76 and motor proteins Myo7a, Klc3 was detected. Remarkably, in the absence of astrocyte activation, we also observed signs of microglial activation at this time point. Both expression of anti-inflammatory cytokines Il13, Ccl24 and pro-inflammatory cytokine receptors Il9r, Il12rb1, Tnfrsf14, Tnfrsf13c, Tnfrsf25, Tnfrsf1b were increased. In the Wnt signaling pathway, we observed increased expression of ligands-receptors Wnt1, Wnt11, Ror2 and also negative regulators Notum, Sfrp5, Sost. RNAseq results after DSN treatment correlated at a high level with RNAseq results without DSN, but there was a proportion of genes that shifted their logFC values. They are mostly rare transcripts TPM 1–10 with higher 0.5–0.9 GC content.

Inference technique for the synaptic conductances in rhythmically active networks and application to respiratory central pattern generation circuits.

Unraveling synaptic interactions between excitatory and inhibitory interneurons within rhythmic neural circuits, such as central pattern generation (CPG) circuits for rhythmic motor behaviors, is critical for deciphering circuit interactions and functional architecture, which is a major problem for understanding how neural circuits operate. Here we present a general method for extracting and separating patterns of inhibitory and excitatory synaptic conductances at high temporal resolution from single neuronal intracellular recordings in rhythmically active networks. These post-synaptic conductances reflect the combined synaptic inputs from the key interacting neuronal populations and can reveal the functional connectome of the active circuits. To illustrate the applicability of our analytic technique, we employ our method to infer the synaptic conductance profiles in identified rhythmically active interneurons within key microcircuits of the mammalian (mature rat) brainstem respiratory CPG and provide a perspective on how our approach can resolve the functional interactions and circuit organization of these interneuron populations. We demonstrate the versatility of our approach, which can be applied to any other rhythmic circuits where conditions allow for neuronal intracellular recordings.

Characterization of brain transduction capability of a BBB-penetrant AAV vector in mice, rats and macaques reveals differences in expression profiles

Different screening methods are being developed to generate adeno-associated viral vectors (AAV) with the ability to bypass the blood-brain barrier (BBB) upon intravenous administration. Recently, the AAV9P31 stood out as the most efficient version among a library of peptide-displaying capsids selected in C57BL/6 mice using RNA-driven biopanning. In this work we have characterized in detail its biodistribution in different mouse strains (C57BL/6 and Balb/c), as well as in Sprague Dawley rats and non-human primates (Macaca fascicularis). Using GFP and NanoLuc reporter genes, we confirmed homogeneous infection and transgene expression across the CNS of mice injected intravenously with AAV9P31. A more restricted pattern was observed upon either intracerebroventricular or intraparenchymal injection. Following intravenous delivery, region- and cell-specific differential patterns of transduction were observed in the mouse brain, including a preferential transduction of astrocytes and neurons in the cerebral cortex and striatum, whereas neurons were the only transduced cell type in subcortical locations across the hippocampus, thalamus, hypothalamus, mesencephalon, brainstem and cerebellum. Furthermore, transduced microglial cells were never found in any CNS location. Peripheral organs transduced upon intravenous administration included lung, liver, peritoneum, heart and skeletal muscle. However, a comparable performance of AAV9P31 to bypass the BBB in rats and macaques was not observed, although a more limited neuronal transduction was found in the brainstem of rats upon intravenous delivery. Finally, intracerebroventricular delivery in macaques resulted in neuronal transduction in cortical, subcortical structures and cerebellum following a patchy pattern. In conclusion, the widespread CNS transduction obtained in mice upon intravenous delivery of AAV9P31 represents a powerful tool for modeling a wide variety of neurological disorders as well as an appealing choice for the evaluation of gene therapy-based therapeutics.

SARS-CoV-2 Spike Protein Enhances Carboxypeptidase Activity of Angiotensin-Converting Enzyme 2.

In this study, we investigated whether severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein may modify angiotensin-converting enzyme 2 (ACE2) activity in the plasma, heart, kidney, liver, lung, and six brain regions (amygdala, brain stem, cortex, hippocampus, hypothalamus, and striatum) of diabetic and hypertensive rats. We determine ACE2 activity in the plasma and lysates of heart, kidney, liver, lung, and six brain regions. MLN-4760 inhibits ACE2 activity in the plasma and all organs. On the other hand, soluble ACE2 (sACE2) activity increased in the plasma of diabetic rats, and there was no change in the plasma of hypertensive rats. ACE2 activity was augmented in the liver, brain stem, and striatum, while it decreased in the kidney, amygdala, cortex, and hippocampus of diabetic rats. ACE2 activity increased in the kidney, liver, and lung, while it decreased in the heart, amygdala, cortex, and hypothalamus of hypertensive rats. We measured the ACE2 content via enzyme-linked immunosorbent assay and found that ACE2 protein levels increased in the heart, while it decreased in the plasma, kidney, brain stem, cortex, hippocampus, hypothalamus, and striatum of diabetic rats. ACE2 protein levels decreased in the brain stem, cortex, hippocampus, and hypothalamus of hypertensive rats. Our data showed that the spike protein enhanced ACE2 activity in the liver and lungs of diabetic rats, as well as in the heart and three of the brain regions (cortex, hypothalamus, and striatum) of hypertensive rats.

Noradrenergic current responses of neurons in rat oculomotor neural integrators.

The prepositus hypoglossi nucleus (PHN) and the interstitial nucleus of Cajal (INC) are involved in the control of horizontal and vertical gaze, respectively. A previous study showed that PHN neurons exhibit depolarized or hyperpolarized responses to noradrenaline (NA). However, the adrenoceptor types that participate in NA-induced responses and the effects of NA on INC neurons have not yet been investigated. Furthermore, the relationship between NA-induced responses and neuron types defined by neurotransmitter phenotypes has not been determined. In this study, we investigated NA-induced current responses in PHN and INC neurons and the relationships between these responses and neuron types using whole cell recordings in wild-type and transgenic rat brainstem slices. Local application of NA to the cell soma induced slow inward (SI) and slow outward (SO) currents that were mainly mediated by α1 and α2 adrenoceptors, respectively. These current responses were observed in both PHN and INC neurons, although the proportion of INC neurons that responded to NA was low. Analyses of the distributions of the current responses revealed that in the PHN, all fluorescently identified inhibitory neurons exhibited SI currents, whereas glutamatergic and cholinergic neurons exhibited both SI and SO currents. In the INC, glutamatergic and inhibitory neurons preferentially exhibited SI and SO currents, respectively. When the PHN and INC neurons were characterized by their firing pattern, we found that the proportions of the currents depended on their firing pattern. These results suggest that various modes of noradrenergic modulation in horizontal and vertical neural integrators are dependent on neuron type.NEW & NOTEWORTHY Noradrenergic modulation of oculomotor neural integrators involved in gaze control has not been elucidated. Here, we report that noradrenaline (NA)-induced slow inward (SI) and outward (SO) currents are mediated mainly by α1 and α2 adrenoceptors in neurons that participate in horizontal and vertical gaze control. The NA-induced current responses differed depending on the neurotransmitter phenotype and firing pattern. These results suggest various modes of noradrenergic modulation in horizontal and vertical integrator neurons.

Serotonin 2C receptor in a rat model of temporal lobe epilepsy: From brainstem expression to pharmacological blockade in relation to ventilatory function.

Because of its involvement in breathing control and neuronal excitability, dysregulation of the serotonin (5-HT) 2C receptor (5-HT2C) might play a key role in sudden unexpected death in epilepsy. Seizure-induced respiratory arrest is thus prevented by a 5-HT2B/C agonist in different seizure model. However, the specific contribution of 5-HT2C in chronic epilepsy-related respiratory dysfunction remains unknown. In a rat model of temporal lobe epilepsy (EPI rats), in which we previously reported interictal respiratory dysfunctions and a reduction of brainstem 5-HT tone, quantitative reverse transcriptase polymerase chain reaction showed overexpression of TPH2 (5-HT synthesis enzyme), SERT (5-HT reuptake transporter), and 5-HT2C transcript levels in the brainstem of EPI rats, and of RNA-specific adenosine deaminase (ADAR1, ADAR2) involved in the production of 5-HT2C isoforms. Interictal ventilation was assessed with whole-body plethysmography before and 2 h after administration of SB242084 (2 mg/kg), a specific antagonist of 5-HT2C. As expected, SB242084 administration induced a progressive decrease in ventilatory parameters and an alteration of breathing stability in both control and EPI rats. However, the size of the SB242084 effect was lower in EPI rats than in controls. Increased 5-HT2C gene expression in the brainstem of EPI rats could be part of a compensatory mechanism against epilepsy-related low 5-HT tone and expression of 5-HT2C isoforms for which 5-HT affinity might be lower.

Annexin A1 conveys neuroprotective function via inhibiting oxidative stress in diffuse axonal injury of rats.

Diffuse axonal injury (DAI) is a critical pathological facet of traumatic brain injury (TBI). Oxidative stress plays a significant role in the progress of DAI. Annexin A1 (AnxA1) has been demonstrated to benefit from recovery of neurofunctional outcomes after TBI. However, whether AnxA1 exhibits neuronal protective function by modulating oxidative stress in DAI remains unknown. Expression of AnxA1 was evaluated via real-time PCR and western blotting in rat brainstem after DAI. The neurological effect of AnxA1 following DAI through quantification of modified neurologic severity score (mNSS) was compared between wild-type and AnxA1-knockout rats. Brain edema and neuronal apoptosis, as well as expression of oxidative factors and inflammatory cytokines, were analyzed between wild-type and AnxA1 deficiency rats after DAI. Furthermore, mNSS, oxidative and inflammatory cytokines were assayed after timely administration of recombinant AnxA1 for DAI rats. In the brainstem of DAI, the expression of AnxA1 remarkably increased. Ablation of AnxA1 increased the mNSS score and brain water content of rats after DAI. Neuron apoptosis in the brainstem after DAI was exaggerated by AnxA1 deficiency. In addition, AnxA1 deficiency significantly upregulated the level of oxidative and inflammatory factors in the brainstem of DAI rats. Moreover, mNSS decreased by AnxA1 treatment in rats following DAI. Expression of oxidative and inflammatory molecules in rat brainstem subjected to DAI inhibited by AnxA1 administration. AnxA1 exhibited neuronal protective function in the progression of DAI mainly dependent on suppressing oxidative stress and inflammation.

Peritoneal sepsis caused by Escherichia coli triggers brainstem inflammation and alters the function of sympatho-respiratory control circuits

BackgroundSepsis has a high mortality rate due to multiple organ failure. However, the influence of peripheral inflammation on brainstem autonomic and respiratory circuits in sepsis is poorly understood. Our working hypothesis is that peripheral inflammation affects central autonomic circuits and consequently contributes to multiorgan failure in sepsis.MethodsIn an Escherichia coli (E. coli)–fibrin clot model of peritonitis, we first recorded ventilatory patterns using plethysmography before and 24 h after fibrin clot implantation. To assess whether peritonitis was associated with brainstem neuro-inflammation, we measured cytokine and chemokine levels in Luminex assays. To determine the effect of E. coli peritonitis on brainstem function, we assessed sympatho-respiratory nerve activities at baseline and during brief (20 s) hypoxemic ischemia challenges using in situ-perfused brainstem preparations (PBPs) from sham or infected rats. PBPs lack peripheral organs and blood, but generate vascular tone and in vivo rhythmic activities in thoracic sympathetic (tSNA), phrenic and vagal nerves.ResultsRespiratory frequency was greater (p < 0.001) at 24 h post-infection with E. coli than in the sham control. However, breath-by-breath variability and total protein in the BALF did not differ. IL-1β (p < 0.05), IL-6 (p < 0.05) and IL-17 (p < 0.04) concentrations were greater in the brainstem of infected rats. In the PBP, integrated tSNA (p < 0.05) and perfusion pressure were greater (p < 0.001), indicating a neural-mediated pathophysiological high sympathetic drive. Moreover, respiratory frequency was greater (p < 0.001) in PBPs from infected rats than from sham rats. Normalized phase durations of inspiration and expiration were greater (p < 0.009, p < 0.015, respectively), but the post-inspiratory phase (p < 0.007) and the breath-by-breath variability (p < 0.001) were less compared to sham PBPs. Hypoxemic ischemia triggered a biphasic response, respiratory augmentation followed by depression. PBPs from infected rats had weaker respiratory augmentation (p < 0.001) and depression (p < 0.001) than PBPs from sham rats. In contrast, tSNA in E. coli-treated PBPs was enhanced throughout the entire response to hypoxemic ischemia (p < 0.01), consistent with sympathetic hyperactivity.ConclusionWe show that peripheral sepsis caused brainstem inflammation and impaired sympatho-respiratory motor control in a single day after infection. We conclude that central sympathetic hyperactivity may impact vital organ systems in sepsis.

Suppression by central adenosine A3 receptors of the cholinergic defense against cardiovascular aberrations of sepsis: role of PI3K/MAPKs/NFκB signaling.

Introduction: Despite the established role of peripheral adenosine receptors in sepsis-induced organ dysfunction, little or no data is available on the interaction of central adenosine receptors with sepsis. The current study tested the hypothesis that central adenosine A3 receptors (A3ARs) modulate the cardiovascular aberrations and neuroinflammation triggered by sepsis and their counteraction by the cholinergic antiinflammatory pathway. Methods: Sepsis was induced by cecal ligation and puncture (CLP) in rats pre-instrumented with femoral and intracisternal (i.c.) catheters for hemodynamic monitoring and central drug administration, respectively. Results: The CLP-induced hypotension, reduction in overall heart rate variability (HRV) and sympathovagal imbalance towards parasympathetic predominance were abolished by i.v. nicotine (100μg/kg) or i.c. VUF5574 (A3AR antagonist, 2µg/rat). In addition, the selective A3AR agonist, 3-iodobenzyl-5'-N-methylcarboxamidoadenosine IB-MECA, 4µg/rat, i.c.) exaggerated the hypotension and cardiac autonomic dysfunction induced by sepsis and opposed the favorable nicotine actions against these septic manifestations. Immunohistochemically, IB-MECA abolished the nicotine-mediated downregulation of NFκB and NOX2 expression in rostral ventrolateral medullary areas (RVLM) of brainstem of septic rats. The inhibitory actions of IB-MECA on nicotine responses disappeared after i.c. administration of PD98059 (MAPK-ERK inhibitor), SP600125 (MAPK-JNK inhibitor) or wortmannin (PI3K inhibitor). Moreover, infliximab (TNFα inhibitor) eliminated the IB-MECA-induced rises in RVLM-NFκB expression and falls in HRV, but not blood pressure. Conclusion: Central PI3K/MAPKs pathway mediates the A3AR counteraction of cholinergic defenses against cardiovascular and neuroinflammatory aberrations in sepsis.

Acute poisoning by chlorpyrifos differentially impacts survival and cardiorespiratory function in normotensive and hypertensive rats

Hypertension is the most important and well-known risk factor for cardiovascular disease (CVD). Recently, acute organophosphate (OP) poisoning has also been pointed as a CVD risk factor. Despite this evidence, no studies have contrasted the acute toxicosis and cardiovascular (CV) effects of OP poisoning under conditions of normotension and hypertension. In this work, adult male normotensive Wistar and Spontaneously Hypertensive rats (SHR) were intraperitoneally injected with saline or chlorpyrifos (CPF), an OP compound, monitored for acute toxicosis signs and 24-h survival. After poisoning, blood pressure, heart rate and ventilation were recorded, the Bezold-Jarisch Reflex (BJR), the Chemoreflex (CR) were chemically activated, as well as the cardiac autonomic tone (AUT) was assessed. Erythrocyte and brainstem acetylcholinesterase and plasmatic butyrylcholinesterase (BuChE) activities were measured as well as lipid peroxidation, advanced oxidation protein products (AOPP), nitrite/nitrate levels, expression of catalase, TNFα and angiotensin-I converting enzyme (ACE-1) within the brainstem. CPF induced a much more pronounced acute toxicosis and 33 % lethality in SHR. CPF poisoning impaired ventilation in SHR, the BJR reflex responses in Wistar rats, and the chemoreflex tachypneic response in both strains. CPF inhibited activity of cholinesterases in both strains, increased AOPP and nitrite/nitrate levels and expression of TNFα and ACE-1 in the brainstem of Wistar rats. Interestingly, SHR presented a reduced intrinsic BuChE activity, an important bioscavenger. Our findings show that, CPF at sublethal doses in normotensive rats lead to lethality and much more pronounced acute toxicity signs in the SHR. We also showed that cardiorespiratory reflexes were differentially impacted after CPF poisoning in both strains and that the cardiorespiratory disfunction seems to be associated with interference in cholinergic transmission, oxidative stress and inflammation. These results points to an increased susceptibility to acute toxicosis in hypertension, which may impose a significant risk to vulnerable populations.

Effect of endogenous substance P on visceral afferent signal integration in the nucleus tractus solitaries of rat brainstem slices

In the first synapse of the blood-pressure-regulating pathway, a neurokinin (NK) family peptide substance P (SP) is release with an excitatory neurotransmitter, glutamate, to enhance the sensitivity of the baroreflex responses. However, the underlying mechanisms of action are not yet well understood. The effects of NK receptor antagonists and agonists on solitary tract stimulation-evoked excitatory postsynaptic responses were recorded using whole-cell patch-clamp recordings of neurons in the medial portion of the nucleus tractus solitarius (mNTS) in the brainstem. SP reduced the amplitude of the evoked excitatory postsynaptic currents (eEPSCs) and shifted the holding current inward, in a dose-dependent manner. The concentrations of SP needed to induce such responses were different between capsaicin-sensitive unmyelinated (C-type) and capsaicin-resistant myelinated (A-type) neurons. The perfusion of a NK1 receptor antagonist, sendide, reduced the amplitude of eEPSCs in all tested neurons but did not affect the levels of the holding current. A Neurokinin type 1 receptor (NK1 receptor) agonist, [Sar9, Met(O2)11]-SP, reduced the amplitude of the eEPSCs and shifted the holding current inward in capsaicin-resistant neurons; however, it failed to induce any significant changes in the capsaicin-sensitive neurons. Furthermore, a selective Neurokinin type 3 receptor (NK3 receptor) antagonist, SB223412, failed to induce any changes in any tested neuron. In current-clamp experiments, sendide reduced solitary tract (ST)-stimulation evoked firing of action potentials in both A- and C-type neurons. [Sar9, Met(O2)11]-SP suppressed the firing of the action potentials in C-type but not A-type neurons. In spontaneous synaptic recordings, SP reduced frequency of the sEPSCs in CAP sensitive neuron but NK1 agonist reduced at capsaicin resistant neurons. Taken together, the findings show that ST activation leads to the co-transmission of SP and glutamate and enhances baroreflex sensitivity by potentiating the amplitude of eEPSC in an NK1 receptor activity-dependent manner.

Neonatal overnutritional programming impairs the hypophagia and neuron activation induced by acute lipopolysaccharide in adult male rats

ABSTRACT Nutritional status during critical windows in early development can challenge metabolic functions and physiological responses to immune stress in adulthood, such as the systemic inflammation induced by lipopolysaccharide (LPS). The aim of this study was to investigate the long-term effects of post-natal over- and undernutrition on the anorexigenic effect of LPS and its association with neuronal activation in the brainstem and hypothalamus of male rats. Animals were raised in litters of 3 (small – SL), 10 (normal – NL), or 16 (large – LL) pups per dam. On post-natal day 60, male rats were treated with LPS (500 µg/Kg) or vehicle for the evaluation of food intake and c-Fos expression in the area postrema (AP), nucleus of solitary tract (NTS), and paraventricular (PVN), arcuate (ARC), ventromedial (VMH), and dorsomedial (DMH) nuclei of the hypothalamus. SL, NL, and LL animals showed a decreased food consumption after LPS treatment. In under- and normonourished animals, peripheral LPS induced an increase in neuronal activation in the brainstem, PaV, PaMP, and ARC and a decrease in the number of c-Fos-ir neurons in the DMH. Overnourished rats showed a reduced hypophagic response, lower neuron activation in the NTS and PaMP, and no response in the DMH induced by LPS. These results indicate that early nutritional programming displays different responses to LPS, by means of neonatal overnutrition decreasing LPS-mediated anorexigenic effect and neuronal activation in the NTS and hypothalamic nuclei.

Cross-frequency coupling between slow harmonics via the real brainstem oscillators: An in vivo animal study.

Brain waves of discrete rhythms (gamma to delta frequency ranges) are ubiquitously recorded and interpreted with respect to probable corresponding specific functions. The most challenging idea of interpreting varied frequencies of brain waves has been postulated as a communication mechanism in which different neuronal assemblies use specific ranges of frequencies cooperatively. One promising candidate is cross-frequency coupling (CFC), in which some neuronal assemblies efficiently utilize the fastest gamma range brain waves as an information carrier (phase-amplitude CFC); however, phase-phase CFC via the slowest delta and theta waves has rarely been described to date. Moreover, CFC has rarely been reported in the animal brainstem including humans, which most likely utilizes the slowest waves (delta and theta ranges). Harmonic waves are characterized by the presence of a fundamental frequency with several overtones, multiples of the fundamental frequency. Rat brainstem waves seemed to consist of slow harmonics with different frequencies that could cooperatively produce a phase-phase CFC. Harmonic rhythms of different frequency ranges can cross-couple with each other to sustain robust and resilient consonance via real oscillators, notwithstanding any perturbations.

Caffeine alleviates anxiety-like behavior and brainstem lesions in a rotenone-induced rat model of Parkinson's disease

BackgroundParkinson's disease (PD) is a neurodegenerative disorder characterized by motor and non-motor symptoms. In 2016, approximately 6.1 million individuals were affected by PD, with 211,296 deaths attributed to the disease. The understanding of PD initially came from the observation of dopaminergic system alterations in a specific region of the brainstem, indicating that the core motor and non-motor features of PD are closely associated with brainstem dysfunction. The primary treatment approach for PD revolves around dopamine replacement, as many of the symptoms are responsive to this therapeutic intervention. However, long-term administration of this approach is linked to several complications, and a definitive gold-standard therapy for PD is yet to be identified. The pharmacological management of PD has been challenging and inconsistent, mainly due to the unclear underlying cause of the disease. This study aims to evaluate the effects of caffeine on the brainstem of rats with PD induced by rotenone. MethodologyFifty adult male Wistar rats weighing between 150 and 200 g were used in this study. The rats were randomly divided into five groups of ten rats each: Vehicle Group, Rotenone-only treated Group (rotenone only treated with 3 mg/kg, intraperitoneal administration [IP]), Preventive Group (caffeine 30 mg/kg + rotenone 3 mg/kg, IP), Curative Group (rotenone 3 mg/kg + caffeine 30 mg/kg, IP), and Caffeine only treated Group (caffeine only treated with 30 mg/kg, IP). The animals underwent neurobehavioral assessments, followed by sacrifice. The brains were then excised, weighed, and processed histologically. Appropriate brain sections were taken and processed. Photomicrographs were obtained, morphometric and statistical analysis was performed using an Omax LED digital ResultsThe results demonstrated a significant (p < 0.05) reduction in body weight and relative brain weight, which were increased by caffeine treatments. Rotenone administration led to histological changes similar to those observed in PD, including neuronal structural derangement, degenerated nerve fibers, loss of myelinated neurons, and Nissl substance, as well as downregulation in the expressions of NRF2 and TH in the midbrain. However, these pathological features were counteracted or ameliorated by caffeine treatment. ConclusionOur study contributes additional evidence to the growing body of research supporting the therapeutic potential of caffeine in Parkinson's disease (PD). The results underscore the neuroprotective properties of caffeine and its capacity to mitigate oxidative stress by modulating TH (tyrosine hydroxylase) and cytoplasmic NRF2 (nuclear factor erythroid 2-related factor 2) in the mesencephalon. These findings suggest that caffeine holds promise as a viable treatment option for PD.

Regulation of tight junction proteins and cell death by peroxisome proliferator-activated receptor γ agonist in brainstem of hypertensive rats.

The decrease in tight junction proteins and their adapter proteins in the hypertensive brain is remarkable. Here, we aimed to investigate tight junction proteins and peroxisome proliferator-activated receptor (PPARγ) activation as well as inflammation factors and cell death proteins in the brainstem of hypertension models, namely spontaneously hypertensive rats (SHR) and borderline hypertensive rats (BHR). At first, SHR and BHR groups were treated with PPARγ agonist, pioglitazone. Then, occludin, claudin-1, claudin-2, claudin-12, ZO-1, and NF-κB p65 gene expression levels; pIKKβ, NF-κB p65, TNF, IL-1β, caspase-3, caspase-9 levels, and PARP-1 cleavage were evaluated. Significantly lower pIKKβ, NF-κB p65, TNF, and IL-1β levels were measured in pioglitazone-treated SHR. Results from this study confirm higher occludin (1.35-fold), claudin-2 (7.45-fold), claudin-12 (1.12-fold), and NF-κB p65 subunit (4.76-fold) expressions in the BHR group when compared to the SHR group. Pioglitazone was found effective in terms of regulating gene expression in SHR. Pioglitazone significantly increased occludin (8.17-fold), claudin-2 (2.41-fold), and claudin-12 (1.85-fold) mRNA levels, which were accompanied by decreased cleaved caspase-3, caspase-9 levels, PARP-1 activation, and proinflammatory factor levels in SHR (p ˂ 0.05). Our work has led us to conclude that alterations in tight junction proteins, particularly occludin, and cell death parameters in the brainstem following PPARγ activation may contribute to neuroprotection in essential hypertension.

Proteomic analysis discovers potential biomarkers of early traumatic axonal injury in the brainstem.

Application of Tandem Mass Tags (TMT)-based LC-MS/MS analysis to screen for differentially expressed proteins (DEPs) in traumatic axonal injury (TAI) of the brainstem and to predict potential biomarkers and key molecular mechanisms of brainstem TAI. A modified impact acceleration injury model was used to establish a brainstem TAI model in Sprague-Dawley rats, and the model was evaluated in terms of both functional changes (vital sign measurements) andstructural changes (HE staining, silver-plating staining and β-APP immunohistochemical staining). TMT combined with LC-MS/MS was used to analyse the DEPs in brainstem tissues from TAI and Sham groups. The biological functions of DEPs and potential molecular mechanisms in the hyperacute phase of TAI were analysed by bioinformatics techniques, and candidate biomarkers were validated using western blotting and immunohistochemistry on brainstem tissues from animal models and humans. Based on the successful establishment of the brainstem TAI model in rats, TMT-based proteomics identified 65 DEPs, and bioinformatics analysis indicated that the hyperacute phase of TAI involves multiple stages of biological processes including inflammation, oxidative stress, energy metabolism, neuronal excitotoxicity and apoptosis. Three DEPs, CBR1, EPHX2 and CYP2U1, were selected as candidate biomarkers and all three proteins were found to be significantly expressed in brainstem tissue 30min-7days after TAI in both animal models and humans. Using TMT combined with LC-MS/MS analysis for proteomic study of early TAI in rat brainstem, we report for the first time that CBR1, EPHX2 and CYP2U1 can be used as biomarkers of early TAI in brainstem by means of western blotting and immunohistochemical staining, compensating for the limitations of silver-plating staining and β-APP immunohistochemical staining, especially in the case of very short survival time after TAI (shorter than 30min). A number of other proteins that also have a potential marker role are also presented, providing new insights into the molecular mechanisms, therapeutic targets and forensic identification of early TAI in brainstem.

Propentofylline Improves Thiol-Based Antioxidant Defenses and Limits Lipid Peroxidation following Gliotoxic Injury in the Rat Brainstem.

Propentofylline (PROP) is a methylated xanthine compound that diminishes the activation of microglial cells and astrocytes, which are neuronal cells strongly associated with many neurodegenerative diseases. Based on previously observed remyelination and neuroprotective effects, PROP has also been proposed to increment antioxidant defenses and to prevent oxidative damage in neural tissues. Since most neurodegenerative processes have free radicals as molecular pathological agents, the aim of this study was to evaluate the antioxidant effects of 12.5 mg·kg-1·day-1 PROP in plasma and the brainstem of Wistar rats exposed to the gliotoxic agent 0.1% ethidium bromide (EB) for 7-31 days. The bulk of the data here demonstrates that, after 7 days of EB treatment, TBARS levels were 2-fold higher in the rat CNS than in control, reaching a maximum of 2.4-fold within 15 days. After 31 days of EB treatment, lipoperoxidation in CNS was still 65% higher than that in the control. Clearly, PROP treatment limited the progression of lipoperoxidation in EB-oxidized CNS: it was, for example, 76% lower than in the EB-treated group after 15 days. Most of these effects were associated with PROP-induced activity of glutathione reductase in the brainstem: the EB + PROP group showed 59% higher GR activity than that of the EB or control groups within 7 days. In summary, aligning with previous studies from our group and with literature about MTXs, we observed that propentofylline (PROP) improved the thiol-based antioxidant defenses in the rat brainstem by the induction of the enzymatic activity of glutathione reductase (GR), which diminished lipid oxidation progression and rebalanced the redox status in the CNS.