P7 Rats Research Articles

Effect of M-Cholinoreceptor Blockade on Somatovisceral Intersystemic Interactions in Newborn Rats after Activation of Cholinoreactive Structures

Interaction of slow-wave rhythmic components of cardiac, respiratory and motor activities was analyzed in non-narcotized neonatal (P1) and early postnatal (P16) rat pups under normal conditions and after activation of cholinoreactive structures. A reversible acetylcholinesterase (AChE) inhibitor physostigmine (eserine) and the M-cholinolytic methacin were used in the experiments. It was established that in intact P1 rats intersystemic interactions are mainly realized via oscillations within the near- and multi-minute ranges. Correlative interactions between the rhythms in the decasecond range that were not involved in integrative processes in P1 rats increased significantly at P16. In rat pups of both ages, changes in the intensity and pattern of motor activity (MA) tended to precede the generation of modulatory oscillations of the respiratory and heart rates. AChE inhibition by physostigmine and subsequent activation of cholinoreactive structures evoked a decrease in the level of intersystemic interactions across all ranges of the modulatory rhythms in rats of both ages. The methacin-evoked blockade of peripheral M-cholinoreceptors (M-ChRs) decreased the level of interactions mediated by the near- and multi-minute rhythms at P1 but augmented them at P16. Physostigmine injection at P1 and P16 exerted no significant effect on cardio-somatomotor interactions, with these interactions in P1 rats being maintained by a wider range of the modulatory rhythms. The same situation was also observed during the physostigmine aftereffect period in all interacting systemic pairs. By contrast, after the blockade of peripheral M-ChRs the existing intersystemic interactions were more difficult to be disrupted in P16 rat pups. The activation of cholinoreactive structures, which occurred against the background of M-ChRs blockade, led to disrupt the coordinating interactions in the “MA–respiratory rate” pair. In P1 rats, these functional relationships did not recover, while in P16 rats they were resumed already during the aftereffect period. The power of modulatory rhythms of activity in the systems studied here was not directly related to intersystemic interactions. Changes in intersystemic interactions were due to heterochrony both in the maturation of these functional systems and the involvement of different cholinoreactive structures in the processes of intersystemic regulation.

Potensi Jus Buah Pepaya (Carica Papaya L.) Mencegah Nefrotoksisitas pada Tikus Wistar yang Terpapar Pb Asetat

Lead is a heavy metal with hazardous toxicity causing dysfunction, especially the kidneys as excretion organs. Pb pollution in Indonesia is mostly derived from transportation emissions such as Tetraetillead, which can change into trietillead, a free radical that can reduce endogenous enzyme activity, one of which is SOD pollution. (Superoxide dismutase) which is in various organs, especially the kidneys. A decrease in SOD activity in the kidneys indicates oxidative stress because the enzyme is unable against superoxide anions and causes renal glomerular damage. Kidney damage can increase serum creatinine levels. Papaya juice contains flavonoids, vitamins C, E, and beta-carotene can prevent nephrotoxicity of Pb. The study aimed to obtain the best dose of papaya juice on the SOD activity of Wistar rats kidney tissue and serum creatinine level that exposed by lead acetate. The experiment was conducted experimentally with Completely Randomized Design, which was divided into 6 treatments, P1, P2, P3, of papaya juice, Lead ace with dose: 3.6; 7.2; 14.4 g / 200g of weight and exposed to Pb acetate 50 mg/kg weight. Treatment P4 (rats were given vitamin E 400 IU/kg weight and exposure to Pb acetate 50 mg/kg weight), P5 (rat exposed Pb acetate 50 mg/kg weight), P6 (normal mice) for 28 days. The results showed that papaya juice was the best dose of 14.4 g / 200g weight because the SOD activity of renal tissue was increased, and serum creatinine level decreased significantly. The conclusion is papaya juice can be used to prevent Pb acetate nephrotoxicity.

NT-4 attenuates neuroinflammation via TrkB/PI3K/FoxO1 pathway after germinal matrix hemorrhage in neonatal rats

BackgroundNeuroinflammation plays an important role in pathogenesis of germinal matrix hemorrhage (GMH). Neurotrophin-4 (NT-4) is a member of the neurotrophin family and interacts with the tropomyosin receptor kinase B (TrkB). NT-4 has been shown to confer neuroprotective effects following cerebral ischemia. We aimed to investigate the neuroprotective function of NT-4-TrkB signaling, as well as its downstream signaling cascade phosphatidylinositol-3-kinases (PI3K)/protein kinase B (Akt)/forkhead box protein O1 (FoxO1), following GMH in neonatal rats.MethodsGMH was induced by intraparenchymal injection of bacterial collagenase (0.3 U) in P7 rat pups. A total of 163 pups were used in this study. Recombinant human NT-4 was administered intranasally at 1 h after the collagenase injection. The selective TrkB antagonist ANA-12, selective PI3K inhibitor LY294002, and FoxO1 activating CRISPR were administered intracerebroventricularly at 24 h prior to NT-4 treatment to investigate the underlying mechanism. Short-term and long-term neurobehavioral assessments, immunofluorescence staining, Nissl’s staining, and Western blot were performed.ResultsExpression of phosphorylated TrkB increased after GMH, reaching the peak level at day 3 after hemorrhage. TrkB receptors were observed on neurons, microglia, and astrocytes. The administration of rh-NT-4 induced phosphorylation of TrkB, expression of PI3K, and phosphorylation of Akt. Meanwhile, it decreased FoxO1 and IL-6 levels. Selective inhibition of TrkB/PI3K/Akt signaling in microglia increased the expression levels of FoxO1 and pro-inflammatory cytokines. FoxO1 activating CRISPR increased the expression of IL-6, suggesting that FoxO1 might be a potential inducer of pro-inflammatory factors. These results suggested that PI3K/Akt/FoxO1 signaling may be the downstream pathway of activation of TrkB. The rat pups treated with rh-NT-4 performed better than vehicle-treated animals in both short-term and long-term behavioral tests.ConclusionThese data showed that rh-NT-4 reduced the expression levels of pro-inflammatory cytokines, improved neurological function, attenuated neuroinflammation, and thereby mitigated post-hemorrhagic hydrocephalus after GMH by TrkB/PI3K/Akt/FoxO1 pathway. These results indicated that rh-NT-4 could be a promising therapeutic strategy to ameliorate neuroinflammation and hydrocephalus after GMH or other similar brain injuries.

Resveratrol Prevents Hyperoxia‐Induced Lung Inflammation

Prolonged oxygen exposure of preterm infants results in the development of bronchopulmonary dysplasia (BPD), a chronic lung disease characterized with lung inflammation, remodeling and airway reactivity. Unfortunately, therapeutic options are limited. Resveratrol is known to have anti‐inflammatory and anti‐oxidant properties in different diseases, however it is not clear about its effects on BPD. In this study we investigated the effects of resveratrol in a rat pup model of BPD. We hypothesize that supplementation of rat pups with resveratrol prevents the lung inflammation induced by hyperoxia. At P5 rat pups were assigned either to room air (21% O2)‐ or hyperoxic (≥95% O2)‐groups and exposed for seven days. During this time, animals were supplemented daily with resveratrol (30 mg/kg of body weight; i.p.) or vehicle. At day P12 animals were euthanized and lungs were harvested, and processed for biochemical and immunohistochemistry studies. Pro‐inflammatory cytokines’ levels (IL‐1β and TNF‐α) were measured in whole lung homogenate using ELISA method. Immunostaining for T‐lymphocyte marker (CD3), B‐lymphocyte marker (CD20) and CD45 was performed on 5 μm lung sections. Levels of IL‐1β and TNF‐α in lung tissue of hyperoxic group of animals were increased significantly (p < 0.01) compared to room air group animals. Supplementation of hyperoxic animals prevented this increase of IL‐1β and TNF‐α, while did not change in room air animals. In hyperoxic animals there was an abundance of CD3, CD20 and CD45 stained cells in the lungs of rat pups exposed to hyperoxia compared to room air exposed pups. Supplementation of hyperoxic animals with resveratrol reduced the number of CD3, CD20 and CD45 stained cells. We conclude that resveratrol prevents lung inflammation induced by neonatal hyperoxia and we speculate that resveratrol might be an effective therapeutic approach to prevent the adverse effects of neonatal hyperoxia.Support or Funding InformationSupported by MEST

Prenatal alcohol exposure in the second trimester-equivalent increases the seizure susceptibility in developing rats.

We have previously reported that prenatal alcohol exposure (PAE) in the 2nd trimester-equivalent of gestation is associated with increased N-methyl-d-aspartate (NMDA)-induced generalized tonic-clonic seizures (GTCSs) prevalence in postpartum developing rats. Whether the 1st trimester-equivalent of gestation is also a vulnerable period for developing GTCSs following PAE is unknown. Here, we investigated the effects of a single episode of PAE at embryonic day 8 (E8, in the 1st trimester-equivalent) or E18 (in the 2nd trimester-equivalent) on NMDA-induced seizures in developing rats at postnatal day 7 (P7, the equivalent of preterm newborns) and P15 (the equivalent of term infants). Pregnant Sprague-Dawley rats were given a single oral dose of ethanol (5g/kg body weight) at E8 or E18 and the postpartum rats were tested for the susceptibility to NMDA-induced seizures at either P7 or P15. NMDA-induced seizures consisted of wild running-like behavior (WRLB), flexion seizures (FSs), clonic seizures (CSs), GTCSs, and tonic seizures (TSs); these seizures were observed in both control-treated and PAE-treated, male and female, P7 and P15 rats. Quantification reveals that the overall prevalence of CSs, GTCSs and TSs occurrence were significantly increased in the E18-PAE group compared to E8-PAE group, adjusting for sex and postnatal day. Furthermore, the overall prevalence of FSs and TSs occurrence was significantly increased in PAE-treated males compared to females, adjusting for embryonic stage and postnatal day. The overall prevalence of WRLB and FSs occurrence was also increased in PAE-P7 rats compared to PAE-P15 rats, adjusting for sex and embryonic stage. We conclude that the susceptibility to develop GTCSs was higher when PAE occurred in the 2nd rather than in the 1st trimester-equivalent of gestation.

SIRT1 Mediates Melatonin's Effects on Microglial Activation in Hypoxia: In Vitro and In Vivo Evidence.

Melatonin exerts direct neuroprotection against cerebral hypoxic damage, but the mechanisms of its action on microglia have been less characterized. Using both in vitro and in vivo models of hypoxia, we here focused on the role played by silent mating type information regulation 2 homolog 1 (SIRT1) in melatonin’s effects on microglia. Viability of rat primary microglia or microglial BV2 cells and SH-SY5Y neurons was significantly reduced after chemical hypoxia with CoCl2 (250 μM for 24 h). Melatonin (1 μM) significantly attenuated CoCl2 toxicity on microglia, an effect prevented by selective SIRT1 inhibitor EX527 (5 μM) and AMP-activated protein kinase (AMPK) inhibitor BML-275 (2 μM). CoCl2 did not modify SIRT1 expression, but prevented nuclear localization, while melatonin appeared to restore it. CoCl2 induced nuclear localization of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-kB), an effect contrasted by melatonin in an EX527-dependent fashion. Treatment of microglia with melatonin attenuated potentiation of neurotoxicity. Common carotid occlusion was performed in p7 rats, followed by intraperitoneal injection of melatonin (10 mg/kg). After 24 h, the number of Iba1+ microglia in the hippocampus of hypoxic rats was significantly increased, an effect not prevented by melatonin. At this time, SIRT1 was only detectable in the amoeboid, Iba1+ microglial population selectively localized in the corpus callosum. In these cells, nuclear localization of SIRT1 was significantly lower in hypoxic animals, an effect prevented by melatonin. NF-kB showed an opposite expression pattern, where nuclear localization in Iba1+ cells was significantly higher in hypoxic, but not in melatonin-treated animals. Our findings provide new evidence for a direct effect of melatonin on hypoxic microglia through SIRT1, which appears as a potential pharmacological target against hypoxic-derived neuronal damage.

Exogenous GM1 Ganglioside Attenuates Ketamine-Induced Neurocognitive Impairment in the Developing Rat Brain

A prolonged exposure to ketamine triggers significant neurodegeneration and long-term neurocognitive deficits in the developing brain. Monosialotetrahexosylganglioside (GM1) can limit the neuronal damage from necrosis and apoptosis in neurodegenerative conditions. We aimed to assess whether GM1 can prevent ketamine-induced developmental neurotoxicity. Postnatal day 7 (P7) rat pups received 5 doses of intraperitoneal ketamine (20 mg/kg per dose) at 90-minute intervals for 6 hours. Cognitive functions, determined by using Morris water maze (MWM) including escape latency (at P32-36) and platform crossing (at P37), were compared among the ketamine-exposed pups treated with or without exogenous GM1 (30 mg/kg; n = 12/group). The effect of GM1 on apoptosis in hippocampus was determined by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining and activated caspase 3 measurement. The hippocampal expression of brain-derived neurotrophic factor (BDNF), along with the phosphorylation of protein kinase B (AKT) and extracellular signal-related kinases 1 and 2 (ERK1/2), was detected by western blotting (n = 6/group). Anti-BDNF antibody (2 μg per rat) administered before GM1 treatment was applied to determine the neuroprotective mechanisms of GM1. The rats receiving ketamine exposure experinced cognitive impairment in MWM test compared to the control rats, indicated by prolonged escape latency at P34 (P = .006), P35 (P = .002), and P36 (P = .005). However, in GM1-pretreated rats, ketamine exposure did not induce prolonged escape latency. The exogenous GM1 increased the platform-crossing times at P37 (3.00 ± 2.22 times vs 5.40 ± 1.53 times, mean ± standard deviation; P = .041) and reduced the hippocampal TUNEL-positive cells and cleaved-caspase 3 expression in ketamine-exposed young rats. Ketamine decreased BDNF expression and phosphorylation of AKT and ERK in the hippocampus, whereas exogenous GM1 blocked these ketamine-caused effects. However, for the ketamine-exposed rat pups receiving exogenous GM1, compared to immunoglobulin Y (IgY) isotype control, the BDNF-neutralizing antibody treatment counteracted the exogenous GM1-induced improvement of the escape latency at P36 (41.32 ± 12.37 seconds vs 25.14 ± 8.97 seconds, mean ± standard deviation; P = .036), platform-crossing times at P37 (2.16 ± 1.12 times vs 3.92 ± 1.97 times, mean ± standard deviation; P < .036), apoptotic activity, as well as AKT and ERK1/2 phosphorylation in the hippocampus of ketamine-challenged young rats. Our data suggest that the exogenous GM1 acts on BDNF signaling pathway to ameliorate the cognitive impairment and hippocampal apoptosis induced by ketamine in young rats. Our study may indicate a potential use of GM1 in preventing the cognitive deficits induced by ketamine in the young per se.

Hemin treatment protects neonatal rats from sevoflurane-induced neurotoxicity via the phosphoinositide 3-kinase/Akt pathway

AimsAnaesthesia-related neurotoxicity in the developing brain is a controversial issue that has recently attracted much attention. Hemin plays a protective role in hypoxic and ischemic brain damage; however, its effects on sevoflurane-induced neurotoxicity remain unclear. Our aim was to investigate the mechanisms of sevoflurane neurotoxicity and potential neuroprotective roles of hemin upon sevoflurane exposure. Main methodsHippocampi were harvested 18 h after sevoflurane exposure. Haem oxygenase 1 (HMOX1), superoxide dismutase 2 (SOD2), discs large MAGUK scaffold protein 4 (DLG4), phosphorylated Akt, Akt, cleaved caspase 3, and neuroglobin were detected by western blotting. A water maze test was used to assess learning and memory ability in P30 rats. Key findingsSevoflurane inhalation increased cleaved caspase 3 levels. Hemin treatment enhanced the antioxidant defence response, protecting rats from oxidative stress injury. Hemin plays its neuroprotective role via phosphoinositide 3-kinase (PI3K)/Akt signalling. A single inhalation of sevoflurane did not affect DLG4 expression, while hemin treatment did. Platform crossing increased in rats treated with hemin as well, which may be related to increased DLG4. Neuroglobin expression was not affected, suggesting that it may act upstream of PI3K/Akt signalling. SignificanceOur study demonstrates that hemin plays a protective role in anaesthesia-induced neurotoxicity by both inhibiting apoptosis via the PI3K/Akt pathway and increasing the expression of antioxidant enzymes, reducing oxidative damage. The results provide mechanistic insight into the effects of sevoflurane anaesthesia on the developing brain and suggest that hemin could help avoid these effects.

Inhibition of calcium-calmodulin-dependent phosphodiesterase (PDE1) suppresses inflammatory responses

A novel, potent, and highly specific inhibitor of calcium-calmodulin-dependent phosphodiesterases (PDE) of the PDE1 family, ITI-214, was used to investigate the role of PDE1 in inflammatory responses. ITI-214 dose-dependently suppressed lipopolysaccharide (LPS)-induced gene expression of pro-inflammatory cytokines in an immortalized murine microglial cell line, BV2 cells. RNA profiling (RNA-Seq) was used to analyze the impact of ITI-214 on the BV2 cell transcriptome in the absence and the presence of LPS. ITI-214 was found to regulate classes of genes that are involved in inflammation and cell migration responses to LPS exposure. The gene expression changes seen with ITI-214 treatment were distinct from those elicited by inhibitors of other PDEs with anti-inflammatory activity (e.g., a PDE4 inhibitor), indicating a distinct mechanism of action for PDE1. Functionally, ITI-214 inhibited ADP-induced migration of BV2 cells through a P2Y12-receptor-dependent pathway, possibly due to increases in the extent of cAMP and VASP phosphorylation downstream of receptor activation. Importantly, this effect was recapitulated in P2 rat microglial cells in vitro, indicating that these pathways are active in native microglial cells. These studies are the first to demonstrate that inhibition of PDE1 exerts anti-inflammatory effects through effects on microglia signaling pathways. The ability of PDE1 inhibitors to prevent or dampen excessive inflammatory responses of BV2 cells and microglia provides a basis for exploring their therapeutic utility in the treatment of neurodegenerative diseases associated with increased inflammation and microglia proliferation such as Parkinson's disease and Alzheimer's disease.

Interaction of GABAA and GABAB antagonists after status epilepticus in immature rats

Among neurotransmitter systems affected by status epilepticus (SE) in adult rats are both GABAergic systems. To analyze possible changes of GABAA and GABAB systems in developing rats lithium-pilocarpine SE was induced at postnatal day 12 (P12). Seizures were elicited by a GABAA antagonist pentylenetetrazol (PTZ) 3, 6, 9, and 13 days after SE (i.e., in P15, P18, P21, and P25 rats), and their possible potentiation by a GABAB receptor antagonist CGP46381 was studied. Pilocarpine was replaced by saline in control animals (lithium-paraldehyde [LiPAR]).Pentylenetetrazol in a dose of 50 mg/kg s.c. elicited generalized seizures in nearly all 15-day-old naive rats and in 40% of 18-day-old ones but not in older animals. After SE, PTZ no longer elicited seizures in these two younger groups, i.e., sensitivity of GABAA system was diminished. The GABAB antagonist exhibited proconvulsant effect in P15 and P18 SE as well as LiPAR rats returning the incidence of PTZ-induced seizures to values of control animals. A decrease in the incidence of minimal clonic seizures was seen in P21 LiPAR animals; these seizures in the oldest group were not affected. Change of the effect from proconvulsant to anticonvulsant (or at least to no action) took place before postnatal day 21.Both SE and LiPAR animals exhibited similar changes but their intensity differed, effects in LiPAR controls were usually more expressed than in SE rats.

The role of the spinal cyclooxygenase (COX) for incisional pain in rats at different developmental stages.

Cyclooxygenase enzymes (COX)-1 and COX-2 are important targets for pain relief after surgery, but the spinal contribution of both isoforms is still unclear, e.g., from a developmental point of view. Here, we studied changes of spinal COX-1 and COX-2 expression and their functional relevance in rats of different ages for pain-related behaviour after incision. Mechanical paw withdrawal thresholds (PWT) were assessed before and after incision and after intrathecal administration (IT) of SC-560 (COX-1 inhibitor) or NS-398 (COX-2 inhibitor) in rats aged 5, 14 and 28days (P5, P14, P28). Furthermore, spinal expressions of COX m-RNA and proteins were investigated. In P5 rats, only IT-administered NS-398 but not SC-560 significantly reversed the decreased PWT after incision. In P14 rats, none of the substance modified PWT, and in P28 rats, only SC-560 increased PWT. Spinal COX-2 mRNA and protein were increased in P5 but not in P14 and P28 rats after incision. Whereas COX-2 is located in spinal neurons, COX-1 is mainly found in spinal microglia cells. Our results demonstrate a possible developmental transition from COX-2 to COX-1 activation. Whereas in adult rats spinal COX-1 but not COX-2 is involved in pain-related behaviour after incision, it seems opposite in P5 rats. Interestingly, in P14, neither COX-1 nor COX-2 seems to play a role. This switch may relate to altered neuronal/microglia activation. Our findings indicate specific mechanisms to pain after incision that are age-dependent and may guide further research improving paediatric pain management. Postoperative pain in pediatric patients after surgery is still poorly controlled; this might contribute to long-lasting alteration in the nociceptive system and prolonged chronic pain. Here we show a possible developmental switch in the COX-dependent pathway for nociceptive spinal transmission that may explain why pain management in young children needs to be related to age-dependent mechanisms.

Onset of neonatal locomotor behavior and the mechanical development of Achilles and tail tendons

Tendon tissue engineering approaches are challenged by a limited understanding of the role mechanical loading plays in normal tendon development. We propose that the increased loading that developing postnatal tendons experience with the onset of locomotor behavior impacts tendon formation. The objective of this study was to assess the onset of spontaneous weight-bearing locomotion in postnatal day (P) 1, 5, and 10 rats, and characterize the relationship between locomotion and the mechanical development of weight-bearing and non-weight-bearing tendons. Movement was video recorded and scored to determine non-weight-bearing, partial weight-bearing, and full weight-bearing locomotor behavior at P1, P5, and P10. Achilles tendons, as weight-bearing tendons, and tail tendons, as non-weight-bearing tendons, were mechanically evaluated. We observed a significant increase in locomotor behavior in P10 rats, compared to P1 and P5. We also found corresponding significant differences in the maximum force, stiffness, displacement at maximum force, and cross-sectional area in Achilles tendons, as a function of postnatal age. However, the maximum stress, strain at maximum stress, and elastic modulus remained constant. Tail tendons of P10 rats had significantly higher maximum force, maximum stress, elastic modulus, and stiffness compared to P5. Our results suggest that the onset of locomotor behavior may be providing the mechanical cues regulating postnatal tendon growth, and their mechanical development may proceed differently in weight-bearing and non-weight-bearing tendons. Further analysis of how this loading affects developing tendons in vivo may inform future engineering approaches aiming to apply such mechanical cues to regulate engineered tendon formation in vitro.

3D cellular reconstruction of cortical glia and parenchymal morphometric analysis from Serial Block-Face Electron Microscopy of juvenile rat

With the rapid evolution in the automation of serial electron microscopy in life sciences, the acquisition of terabyte-sized datasets is becoming increasingly common. High resolution serial block-face imaging (SBEM) of biological tissues offers the opportunity to segment and reconstruct nanoscale structures to reveal spatial features previously inaccessible with simple, single section, two-dimensional images. In particular, we focussed here on glial cells, whose reconstruction efforts in literature are still limited, compared to neurons. We imaged a 750,000 cubic micron volume of the somatosensory cortex from a juvenile P14 rat, with 20 nm accuracy. We recognized a total of 186 cells using their nuclei, and classified them as neuronal or glial based on features of the soma and the processes. We reconstructed for the first time 4 almost complete astrocytes and neurons, 4 complete microglia and 4 complete pericytes, including their intracellular mitochondria, 186 nuclei and 213 myelinated axons. We then performed quantitative analysis on the three-dimensional models. Out of the data that we generated, we observed that neurons have larger nuclei, which correlated with their lesser density, and that astrocytes and pericytes have a higher surface to volume ratio, compared to other cell types. All reconstructed morphologies represent an important resource for computational neuroscientists, as morphological quantitative information can be inferred, to tune simulations that take into account the spatial compartmentalization of the different cell types.

Testing of Hypocholesterolemic Effects of Ledok Added with Seaweed in Vivo

Ledok is a traditional food in Nusa Penida, in form of porridge made from corn, cassava, beans, green leaves vegetables and seasoning without rice. Ledok has the potentials to be developed as a functional food, because it contents sufficient nutrients and bioactive component which is dietary fiber. The obyectives of this research is to discover the hypocholesterolemic effect of ledok added with seaweed in vivo . The experiment is conducted by Completely Randomize Design. The are four treatments which are : PO (rat group without hypercholesterol + standard feed = negative control), P1 (rat group with hypercholesterol + standard feed = positive control), P2 (rat group with hypercholesterol + standard feed + ledok 15 %) and P3 (rat group with hypercholesterol + standard feed+ ledok 30 %). Every group consists of six rats. Analysis are taken before (pretest) and after (posttest) feed treatment on total cholesterol, HDL, LDL, triglyceride of rat blood. The feed treatment is given for 28 days. The research shows that there is a lowering of cholesterol total level, triglyceride and cholesterol LDL of rats blood. In the other hand there is raise in rats blood cholesterol HDL level take place by giving mix of standard feed and ledok added with seaweed. Treatment of ledok by 30 % can lower total cholesterol level, triglyceride and LDL in the following order : 43.09 %, 42.30 % and 37.80 % from pretest level. Meanwhile HDL level of rat blood has a raise of 172,31 % from pretest level. Keywords: ledok , hypocholesterolemic, cholesterol, triglyceride DOI : 10.7176/FSQM/90-05 Publication date :September 30 th 2019

Effects of Methamphetamine Exposure on Fear Learning and Memory in Adult and Adolescent Rats.

Methamphetamine (meth) use is often comorbid with anxiety disorders, with both conditions predominant during adolescence. Conditioned fear extinction is the most widely used model to study the fear learning and regulation that are relevant for anxiety disorders. The present study investigates how meth binge injections or meth self-administration affect subsequent fear conditioning, extinction and retrieval in adult and adolescent rats. In experiment 1, postnatal day 35 (P35-adolescent) and P70 (adult) rats were intraperitoneally injected with increasing doses of meth across 9days. At P50 or P85, they underwent fear conditioning followed by extinction and test. In experiments 2a-c, P35 or P70 rats self-administered meth for 11days then received fear conditioning at P50 or P85, followed by extinction and test. We observed that meth binge exposure caused a significant disruption of extinction retrieval in adult but not adolescent rats. Interestingly, meth self-administration in adolescence or adulthood disrupted acquisition of conditioned freezing in adulthood. Meth self-administration in adolescence did not affect conditioned freezing in adolescence. These results suggest that intraperitoneal injections of high doses of meth and meth self-administration have dissociated effects on fear conditioning and extinction during adulthood, while adolescent fear conditioning and extinction are unaffected.

Context fear learning and renewal of extinguished fear are dissociated in juvenile female rats.

Extinction is the decrease in emotion to a cue that was previously associated with an emotionally significant event. It involves repeated presentation of the cue without any consequences. In adult animals, extinguished fear to a cue can return if the cue is presented in a different environment/context to where extinction occurred, referred to as renewal. We have previously reported that developing female, but not male, rats show renewal. This study investigates whether the ability of developing female rats to show renewal is related to their ability in fear conditioning to the context. Additionally, facilitation of context conditioning by weaning previously shown in male rats was tested in developing female rats. In experiment 1, postnatal day 25 (P25) and P18 female rats showed renewal. P25 rats show more fear overall, suggesting a weaker extinction recall in this age. Experiment 2 tested context- and cue-elicited fear either immediately or 24hr following conditioning. At the immediate test, P18 rats showed less context-fear compared with P25 rats. All rats showed low levels of context-fear at the 24hr test. There were no age differences in cued fear. Weaning at P21 did not affect context or cue memory in P25 female rats. These findings suggest that the ability to form contextual fear memory is unrelated to the expression of renewal in juvenile female rats.

Secukinumab attenuates reactive astrogliosis via IL-17RA/(C/EBPβ)/SIRT1 pathway in a rat model of germinal matrix hemorrhage.

AimsReactive astrogliosis plays a critical role in neurological deficits after germinal matrix hemorrhage (GMH). It has been reported that interleukin‐17A and IL‐17A receptor IL‐17RA/(C/EBPβ)/SIRT1 signaling pathway enhances reactive astrogliosis after brain injuries. We evaluated the effects of secukinumab on reactive astrogliosis in a rat pup model of GMH.MethodsA total of 146 Sprague Dawley P7 rat pups were used. GMH was induced by intraparenchymal injection of collagenase. Secukinumab was administered intranasally 1 hour post‐GMH. C/EBPβ CRISPR or SIRT1 antagonist EX527 was administrated intracerebroventricularly (icv) 48 hours and 1 hour before GMH induction, respectively. Neurobehavior, Western blot, histology, and immunohistochemistry were used to assess treatment regiments in the short term and long term.ResultsThe endogenous IL‐17A, IL‐17RA, C/EBPβ, and GFAP and proliferation marker CyclinD1 were increased, while SIRT1 expression was decreased after GMH. Secukinumab treatment improved neurological deficits, reduced ventriculomegaly, and increased cortical thickness. Additionally, treatment increased SIRT1 expression and lowered proliferation proteins PCNA and CyclinD1 as well as GFAP expression. C/EBPβ CRISPR activation plasmid and EX527 reversed the antireactive astrogliosis effects of secukinumab.ConclusionSecukinumab attenuated reactive astrogliosis and reduced neurological deficits after GMH, partly by regulating IL‐17RA/(C/EBPβ)/SIRT1 pathways. Secukinumab may provide a promising therapeutic strategy for GMH patients.

Role of the ganglioglomerular nerve in response to hypoxia in juvenile rats

The superior cervical ganglion (SCG) sends sympathetic input to the carotid body (CB) via the ganglioglomerular nerve (GGN) in order to modulate carotid sinus nerve (CSN) chemosensory afferent discharge via controlling vascular tone. This chemoafferent discharge is then relayed to the nucleus tractus solitarius (NTS) where the information is processed resulting in a compensatory increase in respiration. Hypoxic gas challenge stimulates the GGN. However, little is known as to the effects of ganglioglomerular nerve transection (GGNx) on carotid body chemosensory afferent response to hypoxia (HX).Our objective was to determine the effect of unilateral GGNx on the ventilatory responses elicited by HX (10% O2, 90% N2) gas challenge (5 episodes of 5 min in duration separated by 15 min) in freely‐moving juvenile (P25) Sprague‐Dawley rats.Male rats postnatal (P) 21 were subjected to sham or unilateral (right or left) GGNx and were allowed 4 days to recover. On the day of the experiment, P25 rats were placed in whole‐body plethysmography chambers and subjected to the hypoxic gas exposure. Ventilatory parameters included frequency of breathing, tidal volume, minute ventilation (= frequency x tidal volume), inspiratory and expiatory times, end inspiratory and expiratory pauses, peak inspiratory and expiratory flows, inspiratory and expiratory drive, EF50 (expiratory flow at 50% expired tidal volume) and rejection index (percentage of abnormal breaths).The HX challenge elicited altered ventilatory responses in sham‐operated rats compared to unilateral GGNx. Rejection Index (%) was diminished in unilateral (left and right) GGNx rats compared to sham, in addition to Rpef in only the right GGNx rats. Peak inspiratory flow (mL/s) was increased in only left GGNx rats, while the peak expiratory flow (mL/s) rates were increased in both the right and left GGNx subjects. Minute ventilation (mL/min) exhibited ventilatory roll‐off in sham animals while no such trend was observed in left GGNx rats. Although peak inspiratory flow, inspiratory drive (mL/s), and expiratory drive (mL/s) reached similar plateaus in right GGNx, the onset of the responses was delayed when compared to their sham counterparts. End expiratory pause (msec) showed a more robust response following hypoxic challenge in right GGNx when viewed against the sham. These findings follow a similar trend to those previously demonstrated from unilateral SCG removal, but still possess characteristics that are unique to the GGN alone. This which suggests the role the SCG has in modulating the hypoxic response is through the GGN and the CB, as well as other yet unknown mechanisms.Support or Funding InformationNIH SPARC 1OT2OD023860‐02 SJLewisThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuroprotective effects of inter-alpha inhibitor proteins after hypoxic-ischemic brain injury in neonatal rats

Hypoxic-ischemic (HI) brain injury is one of the most common neurological problems occurring in the perinatal period. Hypothermia is the only approved intervention for neonatal HI encephalopathy. However, this treatment is only partially protective, has a narrow therapeutic time window after birth and only can be used to treat full-term infants. Consequently, additional therapies are critically needed. Inflammation is an important contributing factor to the evolution of HI brain injury in neonates. Inter-alpha Inhibitor Proteins (IAIPs) are immunomodulatory proteins with anti-inflammatory properties. We have previously shown that IAIPs reduce neuronal cell death and improve behavioral outcomes when given after carotid artery ligation, but before hypoxia in male neonatal rats. The objective of the current study was to investigate the neuroprotective effects of treatment with IAIPs given immediately or 6 h after HI in both male and female neonatal rats. HI was induced with the Rice-Vannucci method in postnatal (P) day 7 rats. After ligation of the right common carotid artery, P7 rats were exposed to 90 min of hypoxia (8% oxygen). Human plasma-derived IAIPs or placebo (phosphate buffered saline) was given at zero, 24, and 48 h after HI. Brains were perfused, weighed and fixed 72 h after HI at P10. In a second, delayed treatment group, the same procedure was followed except that IAIPs or placebo were given at 6, 24 and 48 h after HI. Separate sham-operated, placebo-treated groups were exposed to identical protocols but were not exposed to carotid artery ligation and remained in room air. Rat sex was recorded. The effects of IAIPs on HI brain injury were examined using histopathological scoring and immunohistochemical analyses of the brain and by using infarct volume measurements on frozen tissue of the entire brain hemispheres ipsilateral and contralateral to HI injury. IAIPs given immediately after HI improved (P < 0.050) histopathological brain injury across and within the cingulate, caudate/putamen, thalamus, hippocampus and parietal cortex in males, but not in females. In contrast, IAIPs given immediately after HI reduced (P < 0.050) infarct volumes of the hemispheres ipsilateral to HI injury in similarly both the males and females. Treatment with IAIPs also resulted in higher (P < 0.050) brain weights compared with the placebo-treated HI group, reduced (P < 0.050) neuronal and non-neuronal cell death in the cortex and total hemisphere, and also increased the total area of oligodendrocytes determined by CNPase in the ipsilateral hemisphere and corpus callosum (P < 0.050) of male, but not female subjects exposed to HI. Delayed treatment with IAIPs 6 h after HI did not improve histopathological brain injury in males or females, but resulted in higher (P < 0.050) brain weights compared with the placebo-treated HI males. Therefore, treatment with IAIPs immediately after HI improved brain weights and reduced neuropathological brain injury and cell death in male rats, and reduced infarct volume in both male and female neonatal rats. We conclude that IAIPs exert neuroprotective effects after exposure to HI in neonatal rats and may exhibit some sex-related differential effects.

AQB-565 shows promise in preclinical testing in the model of epileptic spasms during infancy: Head-to-head comparison with ACTH

Epileptic spasms during infancy (infantile spasms) represent a serious treatment and social problem despite their rare occurrence. Current treatments include hormonal therapy (adrenocorticotropin-ACTH or corticosteroids) or vigabatrin (per se or in the combination). These treatments are partially effective and with potentially significant adverse effects. Thus, the search for new effective drugs is warranted. We tested efficacy of a novel fusion peptide AQB-565 developed by Aequus Biopharma in a model of infantile spasms consisting of prenatal exposure to betamethasone and repeated postnatal trigger of spasms with N-methyl-d-aspartic acid (NMDA). AQB-565 molecule includes the first 24 amino acids of ACTH, a ten amino acid linker and a modified melanocyte-stimulating hormone molecule. In contrast to ACTH with almost uniform activity over all peripheral and central melanocortin receptor isoforms, AQB is preferentially active on central melanocortin receptors MC3 and MC4. Here, we used equivalent doses of rat ACTH (full molecule) and AQB-565 and compared their efficacy in a prospective randomized test against of repeated bouts of spasms on postnatal days (P)12, P13 and P15 in the rat model. All doses of ACTH (range 0.02–1.0 mg/kg s.c.) and all doses but one of AQB-565 in the same range suppressed spasms in P15 rats (treatment stopped on P14). There was no dose-dependent effect and both compounds had all-or-none effect that is similar to clinical outcome of hormonal treatment of infantile spasms in children. Thus, AQB-565 may represent a novel treatment of infantile spasms similarly effective as ACTH but with potentially limited side effects.