Sex differences in the structure and function of the vasopressin system in the ventral pallidum are associated with the sex-specific regulation of social play behavior in juvenile rats.

Vasopressin (AVP) regulates various social behaviors, often in sex-specific ways, including social play behavior, a rewarding behavior displayed primarily by juveniles. Here, we examined whether and how AVP acting in the brain's reward system regulates social play behavior in juvenile rats. Specifically, we focused on AVP signaling in the ventral pallidum (VP), a brain region that is a part of the reward system. First, we examined the organization of the VP-AVP system in juvenile rats and found sex differences, with higher density of both AVP-immunoreactive fibers and AVP V1a receptor (V1aR) binding in males compared to females while females show a greater number of V1aR-expressing cells compared to males. We further found that, in both sexes, V1aR-expressing cells co-express a GABA marker to a much greater extent (approx. 10 times) than a marker for glutamate. Next, we examined the functional involvement of V1aR-expressing VP cells in social play behavior. We found that exposure to social play enhanced the proportion of activated V1aR-expressing VP cells in males only. Finally, we showed that infusion of a specific V1aR antagonist into the VP increased social play behaviors in juvenile male rats while decreasing these behaviors in juvenile female rats. Overall, these findings reveal structural and functional sex differences in the AVP-V1aR system in the VP that are associated with the sex-specific regulation of social play behavior.

Preliminary exploration of animal models of congenital choledochal cysts.

BACKGROUND: Various animal models have been used to explore the pathogenesis of choledochal cysts (CCs), but with little convincing results. Current surgical techniques can achieve satisfactory outcomes for treatment of CCs. Consequently, recent studies have focused more on clinical issues rather than basic research. Therefore, we need appropriate animal models to further basic research. AIM: To establish an appropriate animal model that may contribute to the investigation of the pathogenesis of CCs. METHODS: Eighty-four specific pathogen-free female Sprague-Dawley rats were randomly allocated to a surgical group, sham surgical group, or control group. A rat model of CC was established by partial ligation of the bile duct. The reliability of the model was confirmed by measurements of serum biochemical indices, morphology of common bile ducts of the rats as well as molecular biology experiments in rat and human tissues. RESULTS: Dilation classified as mild (diameter, ≥ 1 mm to < 3 mm), moderate (≥ 3 mm to < 10 mm), and severe (≥ 10 mm) was observed in 17, 17, and 2 rats in the surgical group, respectively, while no dilation was observed in the control and sham surgical groups. Serum levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin, and total bile acids were significantly elevated in the surgical group as compared to the control group 7 d after surgery, while direct bilirubin, total bilirubin, and gamma-glutamyltransferase were further increased 14 d after surgery. Most of the biochemical indices gradually decreased to normal ranges 28 d after surgery. The protein expression trend of signal transducer and activator of transcription 3 in rat model was consistent with the human CC tissues. CONCLUSION: The model of partial ligation of the bile duct of juvenile rats could morphologically simulate the cystic or fusiform CC, which may contribute to investigating the pathogenesis of CC.

Differences in the pharmacokinetics and steady-state blood concentrations of orally administered lenvatinib in adult and juvenile rats.

Objective: The aim of this study was to compare the pharmacokinetics and steady-state serum concentrations of lenvatinib in adult and juvenile rats. Experimental study: An ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method was developed to quantify lenvatinib in the serum and liver of rats. Six juvenile and six adult rats in each group were orally administered with a single dose of 7.0 mg/kg lenvatinib suspension for pharmacokinetics. Another 12 juvenile and adult rats were subjected to oral gavage with 7.0 mg/kg lenvatinib once daily for 5 days. Biofluild samples were pre-treated by protein precipitation and sorafenib was used as the internal standard for UPLC-MS analysis. The pharmacokinetic parameters were estimated by compartment and statistical model. The mRNA expression of CYP3A2 and SLC22A1 in liver of adult and juvenile rats was measured by real-time fluorescence quantitative PCR (RT-qPCR). Results: The UPLC-MS method met the requirements for quantitative analysis of lenvatinib in serum and liver. The pharmacokinetic results showed that the mean retention time (MRT(0-∞)) was 19.64 ± 7.64 h and 126.38 ± 130.18 h, with AUC(0-∞) values of 3.97 ± 0.73 µg‧mL-1 h and 5.95 ± 2.27 µg mL-1 h in adult and juvenile rats, respectively. When comparing adult rats (0.35 ± 0.15 µg/mL) to juvenile rats, no significant differences were observed in steady-state serum lenvatinib (0.32 ± 0.11 µg/mL), but a noteworthy decrease to one-third of steady-state liver lenvatinib was observed after multiple oral doses of lenvatinib in juvenile rats. Additional findings revealed that the mRNA expression of CYP3A2 and SLC22A1 was notably increased by 6.86 and 14.67 times, respectively, in juvenile rats compared to adult rats. Conclusion: Juvenile rats exhibit lower levels of lenvatinib in the liver's steady-state, potentially due to the disparity in CYP3A2 mRNA expression. These results imply that the dosage of lenvatinib for pediatric patients may need to be augmented in order to attain the desired clinical outcome.

Learning and metabolic brain differences between juvenile male and female rats in the execution of different training regimes of a spatial memory task.

Spatial memory is responsible for encoding spatial information to form a path, storing this mental representation, and evaluating and recovering spatial configurations to find a target location in the environment. It is mainly supported by the hippocampus and its interaction with other structures, such as the prefrontal cortex, and emerges in rodents around postnatal day (PND) 20. Sex differences in spatial tasks have been found in adults, with a supposedly better performance in males. However, few studies have examined sex differences in orientation throughout postnatal development. This study aimed to analyse the performance of juvenile (PND 23) male (n = 18) and female (n = 21) Wistar rats in a spatial reference memory task in the Morris water maze (MWM) with two different training regimes in the acquisition phase, and their subjacent metabolic brain activity. Based on sex, subjects were assigned to two different groups: one that performed four learning trials per day (n = 9 males and n = 8 females) and the other that was submitted to two trials per day (n = 9 males and n = 13 females). After the behavioural protocols, metabolic activity was evaluated using cytochrome c oxidase histochemistry. Results showed no metabolic brain or behavioural differences in the four-trial protocol performance, in which both sexes reached the learning criterion on the fourth day. By contrast, the two-trial protocol revealed an advantage for females, who reached the learning criterion on day four, whereas males needed more training and succeeded on day six. The female group showed lower metabolic activity than the male group in the cingulate and prelimbic cortex. These results suggest a faster consolidation process in the female group than the male group. Further research is needed to understand sex differences in spatial memory at early stages.

Loss of ganglioglomerular nerve input to the carotid body impacts the hypoxic ventilatory response in freely-moving rats.

The carotid bodies are the primary sensors of blood pH, pO2 and pCO2. The ganglioglomerular nerve (GGN) provides post-ganglionic sympathetic nerve input to the carotid bodies, however the physiological relevance of this innervation is still unclear. The main objective of this study was to determine how the absence of the GGN influences the hypoxic ventilatory response in juvenile rats. As such, we determined the ventilatory responses that occur during and following five successive episodes of hypoxic gas challenge (HXC, 10% O2, 90% N2), each separated by 15 min of room-air, in juvenile (P25) sham-operated (SHAM) male Sprague Dawley rats and in those with bilateral transection of the ganglioglomerular nerves (GGNX). The key findings were that 1) resting ventilatory parameters were similar in SHAM and GGNX rats, 2) the initial changes in frequency of breathing, tidal volume, minute ventilation, inspiratory time, peak inspiratory and expiratory flows, and inspiratory and expiratory drives were markedly different in GGNX rats, 3) the initial changes in expiratory time, relaxation time, end inspiratory or expiratory pauses, apneic pause and non-eupneic breathing index (NEBI) were similar in SHAM and GGNX rats, 4) the plateau phases obtained during each HXC were similar in SHAM and GGNX rats, and 5) the ventilatory responses that occurred upon return to room-air were similar in SHAM and GGNX rats. Overall, these changes in ventilation during and following HXC in GGNX rats raises the possibility the loss of GGN input to the carotid bodies effects how primary glomus cells respond to hypoxia and the return to room-air.

Ipsilateral auditory cortex responses to the intact ear after unilateral noise trauma in juvenile rats.

BACKGROUND: While ear stimulation produces a robust response in the contralateral auditory cortex (AC), it produces only a weak response in the ipsilateral AC, known as interhemispheric asymmetry. Unilateral deafness can lead to AC plastic changes, resulting in reduced interhemispheric asymmetry and auditory perceptual consequences. However, the unilateral hearing loss-associated plastic changes are far from fully understood. The purpose of this study was to investigate AC responses to the ipsilateral unimpaired ear after noise injury to the contralateral ear in juvenile rats. METHODS: Rats (50 days) were monaurally exposed to an intense noise (10.0-12.5 kHz, 126 dB SPL) for 2 hours. The unexposed ear-induced ipsilateral AC responses were recorded 2 days and 4 months after exposure and compared between groups. RESULTS: The noise exposure resulted in complete hearing loss in the exposed ear, but normal function in the other. Two days after exposure, the ipsilateral AC response induced by the intact ear was significantly enhanced and the threshold decreased (the early-onset effect). Four months after noise exposure, in addition to the increased response amplitude, the "slow-increasing" firing pattern of the neurons in the ipsilateral AC turned into the contralateral-AC-response-like "sharp-increasing" pattern (the late-onset effect) with shortened response latency. DISCUSSION: The early-onset effect can result from release of inhibition due to decreased contralateral input, while the late-onset effect may imply the formation of direct connections in the ipsilateral auditory pathway. The enhanced AC response may help maintain loudness perception and monaural sound localization after unilateral deafness.

Dezocine relieves the postoperative hyperalgesia in rats through suppressing the hyper-action of Akt1/GSK-3ß pathway.

The relieving role of dezocine in pain after surgery was previously reported, while the potential mechanism was not completely clear. Therefore, the current research probed into the regulatory mechanism of dezocine in pain after surgery. A postoperative pain model was established by performing plantar incision surgery on the juvenile Sprague-Dawley rats. After the rats were treated with dezocine or SC79 (Akt1 activator), the paw withdrawal threshold and paw withdrawal latency of rats were detected to evaluate the mechanical allodynia and thermal hyperalgesia. After the plantar tissue, dorsal root ganglions, and spinal cord of rats were collected, the expressions of Akt1, p-Akt1, GSK-3ß, and p-GSK-3ß in the tissues were determined by western blot to evaluate the activation state of the Akt1/GSK-3ß pathway. After surgery, the paw withdrawal threshold and paw withdrawal latency of rats were lessened, whereas the ratios of p-Akt1/Akt1 and p-GSK-3ß/GSK-3ß were augmented in rat plantar tissue, dorsal root ganglions, and spinal cord. After treatment with dezocine alone, the paw withdrawal threshold and paw withdrawal latency of postoperative rats were elevated, but ratios of p-Akt1/Akt1 and p-GSK-3ß/GSK-3ß were reduced. After co-treatment with dezocine and SC79, SC79 reversed the effects of dezocine on elevating the paw withdrawal threshold and paw withdrawal latency, and reducing the ratios of p-Akt1/Akt1 and p-GSK-3ß/GSK-3ß in postoperative rats. Dezocine ameliorated the postoperative hyperalgesia in rats via repressing the hyper-action of Akt1/GSK-3ß pathway.

Comparative study on detectability of learning and memory disorder between two water maze tests commonly used in juvenile rat toxicity studies using isoflurane inhaled rat model.

Evaluation of learning and memory is crucial in juvenile animal toxicity studies (JAS) during the development of CNS active drugs, but there are no currently recommended test methods. We compared the ability of the Morris water maze (MWM) and the Biel water maze (BWM) to detect learning and memory disorder (LMD) using rats inhaled isoflurane (IFN). Rats were treated with 1% IFN using inhalation on postnatal day (PND) 7 for 6 h. All rats were subjected to the MWM on PND 33 and the BWM on PND 55/57 (Experiment 1), or the BWM on PND 32/33 and the MWM on PND 54/55 (Experiment 2). On PND 70, the brain was weighed and then neurohistopathology was conducted. There were no IFN-related changes in clinical signs and body weight. In the tests beginning on PND 32/33, the MWM clearly detected IFN-related LMD in both sexes whereas the BWM detected LMD only in males. With an additional benefit of a simpler procedure, the MWM was considered superior to the BMW for JAS. LMD was not detected in both mazes tested from PND 54/55/57, which was considered due to weak effect and/or recovery of brain function with growth. Single IFN inhalation on PND 7 was considered useful as positive control to induce LMD caused by postnatal exposure in rats, but stronger treatment regimens was recommended.

Early administration of milrinone ameliorates lung and kidney injury during sepsis in juvenile rats.

BACKGROUND: A sepsis model was created, induced by cecal ligation and puncture (CLP), in juvenile rat groups. Milrinone (MIL), which is known to have a modulatory effect on pro-inflammatory cytokines, was administered to the designated rat groups in the early period before severe sepsis developed. The study was aimed at investigating the possible protective effects of milrinone on the lung and kidney tissues of rats in the late phase of sepsis. METHODS: The rat pups were divided into seven groups with six animals in each group: (1) healthy rats who received no drug; (2) CLP-S12 (sacrificed at hour 12); (3) CLP-S24 (sacrificed at hour 24); (4) CLP-MIL1-S12 (administered with 0.5 mg/kg milrinone at hour 1 and sacrificed at hour 12); (5) CLP-MIL1-S24 (administered with 0.5 mg/kg milrinone at hour 1 and sacrificed at hour 24): (6) CLP-MIL12-S24 (administered with 0.5 mg/kg milrinone at hour 12 and sacrificed at hour 24), (7) and CLP-MIL1,12-S24 (administered with 0.5 mg/kg milrinone at hours 1 and 12 and sacrificed at hour 24). RESULTS: Significant differences were found between the early and late administration of milrinone in terms of both molecular and histopathological results. The results showed that the tissues were significantly preserved in the groups in which milrinone had been started in the early period compared to the sepsis control groups and the groups in which milrinone had been started in the late period. CONCLUSIONS: In addition to the positive inotropic effects of milrinone, its immunomodulatory properties that result in decreased cytokine storm can be beneficial during early period of sepsis.

Oxandrolone treatment in juvenile rats induced anxiety-like behavior in young adult animals.

AIMS: Oxandrolone (OXA) is a synthetic steroid used for the treatment of clinical conditions associated with catabolic states in humans, including children. However, its behavioral effects are not well known. Our goal was to evaluate the anxiety-like behavior induced in young adult rats after the treatment of juvenile animals with OXA. METHODS: Four-week-old male rats were separated into three groups: Control (CON), therapeutic-like OXA dose (TD), and excessive OXA dose (ED), in which 2.5 and 37.5 mg/kg/day of OXA were administered via gavage for four weeks for TD and ED, respectively. Behavior was evaluated through the elevated plus maze (EPM) and open field (OF) tests. Protein expression of catalase (CAT), superoxide dismutase (SOD), Tumor necrosis factor-α (TNF-α), and dopamine receptor 2 (DrD2) were analyzed in tissue samples of the hippocampus, amygdala, and prefrontal cortex by Western Blot. RESULTS: OXA induced anxiety-like behaviors in both TD and ED animals; it decreased the time spent in the open arms of the EPM in both groups and reduced the time spent in the central zone of the OF in the TD group. In the hippocampus, CAT expression was higher in TD compared with both control and ED animals. No differences were found in the amygdala and prefrontal cortex. TNF-α, SOD, and DrD2 levels were not altered in any of the assessed areas. CONCLUSIONS: Treatment of juvenile rats with OXA led to anxiety-like behavior in young adult animals regardless of the dose used, with minor changes in the antioxidant machinery located in the hippocampus.

A Histological and Morphometric Assessment of the Adult and Juvenile Rat Livers after Mild Traumatic Brain Injury.

Traumatic brain injury (TBI) is one of the most severe problems of modern medicine that plays a dominant role in morbidity and mortality in economically developed countries. Our experimental study aimed to evaluate the histological and morphological changes occurring in the liver of adult and juvenile mildly traumatized rats (mTBI) in a time-dependent model. The experiment was performed on 70 adult white rats at three months of age and 70 juvenile rats aged 20 days. The mTBI was modelled by the Impact-Acceleration Model-free fall of weight in the parieto-occipital area. For histopathological comparison, the samples were taken on the 1st, 3rd, 5th, 7th, 14th, and 21st days after TBI. In adult rats, dominated changes in the microcirculatory bed in the form of blood stasis in sinusoidal capillaries and veins, RBC sludge, and adherence to the vessel wall with the subsequent appearance of perivascular and focal leukocytic infiltrates. In juvenile rats, changes in the parenchyma in the form of hepatocyte dystrophy prevailed. In both groups, the highest manifestation of the changes was observed on 5-7 days of the study. On 14-21 days, compensatory phenomena prevailed in both groups. Mild TBI causes changes in the liver of both adult and juvenile rats. The morphological pattern and dynamics of liver changes, due to mild TBI, are different in adult and juvenile rats.

Glyphosate exposure induces synaptic impairment in hippocampal neurons and cognitive deficits in developing rats.

Glyphosate is the active ingredient of several widely used herbicide formulations. Studies based on Glyphosate exposure in different experimental models have suggested that the nervous system represented a key target for its toxicity. Previously, we demonstrated that exposure to glyphosate during gestation induces deficits on behavioral and cognitive function in rats. The aim of the present work was to examine whether cognitive dysfunction induced by Glyphosate was connected to changes on synapse formation and maturation. To understand how glyphosate affects synaptic assembly, we performed in vitro assays on cultured hippocampal neurons that were exposed to the herbicide (0.5 or 1 mg/mL) for 5 or 10 days. Biochemical and immunocytochemical approaches revealed that Glyphosate treated neurons showed a decrease on dendritic complexity and synaptic spine formation and maturation. Moreover, results indicated that Glyphosate decreased synapse formation in hippocampal neurons. To evaluate these effects in vivo, pup rats were treated with 35 or 70 mg/kg of Glyphosate from PND 7 to PND 27, every 48 h. Results indicated that Glyphosate postnatal exposure induced cognitive impairments, since recognition and spatial memory were altered. To go further, we evaluated synaptic protein expression and synaptic organization in hippocampus. Images revealed that Glyphosate treatment downregulates synapsin-1, PSD-95, and CaMKII expression, and also decreased PSD-95 clustering in hippocampus. Taken together, these findings demonstrate for the first time that Glyphosate exposure affects synaptic assembly and reduced synaptic protein expression in hippocampus and that likely triggers the impairment of cognitive function and neuronal connectivity.

Biodistribution, cardiac and neurobehavioral assessments, and neurotransmitter quantification in juvenile rats following oral administration of aluminum oxide nanoparticles.

Little is known about the uptake, biodistribution, and biological responses of nanoparticles (NPs) and their toxicity in developing animals. Here, male and female juvenile Sprague-Dawley rats received four consecutive daily doses of 10 mg/kg Al2 O3 NP (diameter: 24 nm [transmission electron microscope], hydrodynamic diameter: 148 nm) or vehicle control (water) by gavage between postnatal days (PNDs) 17-20. Basic neurobehavioral and cardiac assessments were performed on PND 20. Animals were sacrificed on PND 21, and selected tissues were collected, weighed, and processed for histopathology or neurotransmitter analysis. The biodistribution of Al2 O3 NP in tissue sections of the intestine, liver, spleen, kidney, and lymph nodes were evaluated using enhanced dark-field microscopy (EDM) and hyperspectral imaging (HSI). Liver-to-body weight ratio was significantly increased for male pups administered Al2 O3 NP compared with control. HSI suggested that Al2 O3 NP was more abundant in the duodenum and ileum tissue of the female pups compared with the male pups, whereas the abundance of NP was similar for males and females in the other tissues. The abundance of NP was higher in the liver compared with spleen, lymph nodes, and kidney. Homovanillic acid and norepinephrine concentrations in brain were significantly decreased following Al2 O3 NP administration in female and male pups, whereas 5-hydroxyindoleacetic acid was significantly increased in male pups. EDM/HSI indicates intestinal uptake of Al2 O3 NP following oral administration. Al2 O3 NP altered neurotransmitter/metabolite concentrations in juvenile rats' brain tissues. Together, these data suggest that orally administered Al2 O3 NP interferes with the brain biochemistry in both female and male pups.

Effects of prenatal ethanol exposure on acoustic characteristics of play fighting-induced ultrasonic vocalizations in juvenile rats.

Juvenile rats display rough-and-tumble playing with conspecifics (play fighting behavior) and produce 22 and 50 kHz ultrasonic vocalizations (USVs). The 22 kHz USV is considered to reflect negative emotionality such as anxiety, fear, and distress, whereas the 50 kHz USV is considered to reflect positive emotionality such as joy, happiness, and satisfaction. USV is a sensitive tool for measuring emotionality in socially interactive situations. However, effects of prenatal ethanol-exposure on the acoustic characteristics of play fighting-induced USVs have remained unclear. In Experiment I, we recorded USVs produced by prenatally ethanol-exposed rats during play fighting on postnatal days (PNDs) 40-42 and examined the acoustic characteristics of negative and positive emotion-induced USVs. In Experiment II, we examined the anxiety levels through elevated plus maze testing on PNDs 37-39 and frequencies of playful attacks on PNDs 43-45 in ethanol-exposed rats. Ethanol was administered to pregnant rats in three gradually increased concentrations between gestational days (GDs) 8 and 20. From GDs 14 to 20, ethanol-containing tap water at concentrations of 30% and 15% (v/v) was administered to the high- and low-ethanol groups, respectively. Tap water without added ethanol was given to the control group. On PNDs 40-42, three rats from the same sex and same ethanol concentration group but from different litters were placed together into a playing cage for play fighting. The high-ethanol male triads displayed elevations of 20-35 kHz USVs reflecting negative emotionality and reductions of 45-70 kHz USVs reflecting positive emotionality compared with both the low-ethanol and control male triads. The high-ethanol male triads had prominent elevations of 20-35 kHz USVs with durations longer than 200 ms, whereas the control male triads did not produce such 20-35 kHz USVs at all. There was no difference in USV acoustic characteristics among the female triads. In addition, the high-ethanol male rats exhibited greater anxiety levels and less frequencies of play fighting than the control male rats. Altogether, we conclude that prenatal exposure to ethanol enhances negative emotionality such as anxiety and, accordingly, 20-35 kHz USVs reflecting negative emotionality are produced with a marked decrease in play fighting, suggesting difficulties in social interactions with conspecifics.

Comparative pharmacokinetics of baicalin and geniposide in juvenile and adult rats after oral administration of Qingkailing Granules.

Objective: To explore the effect of age on Qingkailing Granules disposition by comparing the pharmacokinetics of geniposide and baicalin in juvenile and adult rats. Methods: A simple and rapid LC-MS/MS method was developed and validated to simultaneously determine geniposide and baicalin in rat plasma after a simple protein precipitation. The analytes were separated on an Agilent ZORBAX Extend-C18 column. The mobile phase consisted of acetonitrile and water with 0.1% (volume percent) formic acid at a flow rate of 0.6 mL/min. The ionization was conducted using an ESI source in negative ion mode. Multiple reaction monitoring was used for quantification at transitions of m/z 445.0 â†’ m/z 268.9 for baicalin, m/z 433.2 â†’ m/z 225.0 for geniposide, m/z 431.0 â†’ m/z 341.0 for vitexin (IS). Juvenile and adult rats were administrated Qingkailing Granules (3 g/kg) orally. Plasma concentrations of baicalin and geniposide were determined by LC-MS/MS. Results: The linear ranges of the analytes were 1-1000 ng/mL for baicalin and 2-2000 ng/mL for geniposide. The method was successfully applied to compare the pharmacokinetics of the analytes between juvenile and adult rats after oral administration of Qingkailing Granules. AUC was bigger in adult rats, while t 1/2 was longer in juvenile rats. Conclusion: These results suggested that the absorption and elimination of baicalin and geniposide in juvenile rats was lower than that in adult rats. Additional attention should be paid to the pharmacokinetic difference when Qingkailing Granules were used in children.

Hippocampal astrocytes in juvenile rats with persistent epilepsy: The role of cannabinoid receptor type 2 in regulating MAPK pathway / 中国组织工程研究

BACKGROUND: Astrocyte proliferation is an important morphological change in epilepsy. Proliferated glial cells can produce cytokines, and in turn activates JAK/STAT signal transduction to promote glial cell proliferation, which affects the occurrence and recurrence of epilepsy. Astrocytes and signal transduction pathways interact with each other to play a role in the pathogenesis of epilepsy. OBJECTIVE: To investigate the effect of cannabinoid receptor type 2 (CB2R) on the activation of ERK, p38, and JNK proteins in astrocytes and MAPK pathways in juvenile rats with persistent epilepsy. METHODS: Forty healthy male Sprague-Dawley rats (18-21 days old) were randomly divided into four groups: normal control group, epilepsy model group, CB2R agonist JWH133 group, CB2R antagonist AM630 group. The normal control group was given only normal saline. In the other groups, rats were intraperitoneally injected with lithium chloride and pilocarpine to establish epilepsy models, and different interventions were performed. Twenty-four hours after the onset of epilepsy, brain tissues were taken. Co-expression of GFAP and p-ERK, p-p38, and p-JNK in hippocampal tissue was detected by immunofluorescence. Real-time PCR was used to detect the expression of GFAP mRNA in hippocampal tissue. RESULTS AND CONCLUSION: The co-expression of GFAP/p-ERK and GFAP/p-p38 was significantly higher in the epilepsy model group than the normal control group (P < 0.05), significantly lower in the JWH133 group than the epilepsy model group (P < 0.05), and significantly higher in the AM630 group than the JWH133 group (P < 0.05). The co-expression of GFAP/p-JNK was significantly lower in the epilepsy model group than in normal control group (P < 0.05), significantly higher in the JWH133 group than the epilepsy model group (P < 0.05), and significantly lower in the AM630 group than the JWH133 group (P < 0.05). The mRNA expression of GFAP was significantly decreased in the epilepsy model group compared with the normal control group (P < 0.05), significantly increased in the JWH133 group compared with the epilepsy model group (P < 0.05), and significantly reduced in the AM630 group compared with the JWH133 group (P < 0.05). Therefore, CB2R can regulate the expression of ERK, p38, JNK proteins in the MAPK pathway, thereby affecting astrocytes in the hippocampus of juvenile rats with persistent epilepsy.

Comparative pharmacokinetics of baicalin and geniposide in juvenile and adult rats after oral administration of Qingkailing Granules / 中草药·英文版

Objective: To explore the effect of age on Qingkailing Granules disposition by comparing the pharmacokinetics of geniposide and baicalin in juvenile and adult rats. Methods: A simple and rapid LC-MS/MS method was developed and validated to simultaneously determine geniposide and baicalin in rat plasma after a simple protein precipitation. The analytes were separated on an Agilent ZORBAX Extend-C18 column. The mobile phase consisted of acetonitrile and water with 0.1% (volume percent) formic acid at a flow rate of 0.6 mL/min. The ionization was conducted using an ESI source in negative ion mode. Multiple reaction monitoring was used for quantification at transitions of m/z 445.0 → m/z 268.9 for baicalin, m/z 433.2 → m/z 225.0 for geniposide, m/z 431.0 → m/z 341.0 for vitexin (IS). Juvenile and adult rats were administrated Qingkailing Granules (3 g/kg) orally. Plasma concentrations of baicalin and geniposide were determined by LC-MS/MS. Results: The linear ranges of the analytes were 1–1000 ng/mL for baicalin and 2–2000 ng/mL for geniposide. The method was successfully applied to compare the pharmacokinetics of the analytes between juvenile and adult rats after oral administration of Qingkailing Granules. AUC was bigger in adult rats, while t1/2 was longer in juvenile rats. Conclusion: These results suggested that the absorption and elimination of baicalin and geniposide in juvenile rats was lower than that in adult rats. Additional attention should be paid to the pharmacokinetic difference when Qingkailing Granules were used in children.

Long-term impairment of social behavior, vocalizations and motor activity induced by bilateral lesions of the fastigial nucleus in juvenile rats.

The cerebellum is increasingly recognized to be involved in limbic and cognitive-associative functioning. Cerebellar cognitive affective syndromes may result from various types of injuries. Cerebellar mutism may occur in children after resection of midline tumors in the posterior fossa, which has been thought to be related to damage to the cerebellar vermis. Here, we investigated whether bilateral lesions of the fastigial nucleus, which is located within the upper vermis, would affect social behavior in a rat model. Juvenile male Sprague-Dawley rats, aged 23 days, underwent bilateral thermocoagulation of the fastigial nucleus via stereotaxically implanted electrodes under general anesthesia. Electrodes were inserted without application of electric current in a sham-lesion group and naïve rats served as additional controls. All groups underwent standardized examination before surgery and on specific time points up to 49 days after surgery to investigate locomotor activity, motor coordination, social behavior, and ultrasound vocalizations during social interaction. Finally, lesions were verified histologically. Playing behavior and vocalizations were reduced up to 4 weeks after surgery in rats of the lesion group compared to rats with sham-lesions and controls. After surgery in rats of the lesion group, locomotor activity was disturbed for 3 days as compared to sham-lesion rats, but for 4 weeks as compared to controls. Motor coordination measured by the rotarod and balance beam test was compromised until adulthood. Bilateral lesions of the fastigial nucleus in juvenile rats cause a severe and long-lasting reduction of social interaction and motor coordination in juvenile rats, which has some similarities to cerebellar cognitive affective syndromes in the human context. This indicates a modulating role of the fastigial nucleus with regard to neural circuitries relevant for social behavior, such as the limbic system and the prefrontal cortex.

Enhanced setup for wired continuous long-term EEG monitoring in juvenile and adult rats: application for epilepsy and other disorders.

BACKGROUND: The electroencephalogram (EEG) is a widely used laboratory technique in rodent models of epilepsy, traumatic brain injury (TBI), and other neurological diseases accompanied by seizures. Obtaining prolonged continuous EEG tracings over weeks to months is essential to adequately answer research questions related to the chronobiology of seizure emergence, and to the effect of potential novel treatment strategies. Current EEG recording methods include wired and the more recent but very costly wireless technologies. Wired continuous long-term EEG in rodents remains the mainstay approach but is often technically challenging due to the notorious frequent EEG cable disconnections from the rodent's head, and to poor signal-to-noise ratio especially when simultaneously monitoring multiple animals. Premature EEG cable disconnections and cable movement-related artifacts result from the animal's natural mobility, and subsequent tension on the EEG wires, as well as from potential vigorous and frequent seizures. These challenges are often accompanied by injuries to the scalp, and result in early terminations of costly experiments. RESULTS: Here we describe an enhanced customized swivel-balance EEG-cage system that allows tension-free rat mobility. The cage setup markedly improves the safety and longevity of current existing wired continuous long-term EEG. Prevention of EEG cable detachments is further enhanced by a special attention to surgical electrode anchoring to the skull. In addition to mechanically preventing premature disconnections, the detailed stepwise approach to the electrical shielding, wiring and grounding required for artifact-free high signal-to-noise ratio recordings is also included. The successful application of our EEG cage system in various rat models of brain insults and epilepsy is described with illustrative high quality tracings of seizures and electrographic patterns obtained during continuous and simultaneous monitoring of multiple rats early and up to 3 months post-brain insult. CONCLUSION: Our simple-to-implement key modifications to the EEG cage setup allow the safe acquisition of substantial high quality wired EEG data without resorting to the still costly wireless technologies.

Juvenile rat and pediatric trazodone studies: how to gain extra sensitivity to overcome bioanalytical challenges.

AIM: Trazodone (TZD) is used for the treatment of depression in adults and, off-label, as a sleep medication in adult and pediatric populations. The off-label use is well documented, however further clinical studies are needed to confirm its efficacy and safety for the treatment of sleep disorders. In this scenario, we developed a bioanalytical method to quantify low TZD concentrations in samples collected by capillary microsampling (CMS) to support dose finding, Good Laboratory Practice juvenile rat toxicokinetic and upcoming pediatric studies. METHODOLOGY: A method using only 8 µl of plasma was developed and successfully used for analyzing CMS samples from juvenile rats throughout toxicokinetic study. CONCLUSION: By harmoniously maximizing each analytical step, we achieved a sensitive method to quantify TZD in CMS samples.