Ketamine-induced Neuronal Apoptosis Research Articles

Integrin-dependent microgliosis mediates ketamine-induced neuronal apoptosis during postnatal rat retinal development

PurposeRemodeling of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) plays a pivotal role for microglia in developing retina. We tested whether integrin-dependent microgliosis mediates ketamine-induced neuronal apoptosis in the developing rat retina. MethodsWe performed immunofluorescence assays to investigate the role of integrin receptors expressed in the microglia in ketamine-induced neuronal apoptosis. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to investigate the protein and mRNA levels of cytokines (TNF-α, IL-1β) and/or chemokines (CCL2, CXCL6, CXCL10, and CXCL12). Experiments were performed using whole-mount retinas dissected from P7 Sprague-Dawley rats. ResultsIntegrin receptors expressed in microglia were upregulated in ketamine-induced neuronal apoptosis in the early developing rat retina. Downregulating integrin receptors with RGD peptide ameliorated ketamine-induced microgliosis through: 1) ameliorating the change in microglia morphology from immature ramified microglia to an amoeboid state; 2) decreasing the number of microglia and intensity of activated microglia in the retinal ganglion cell layer (GCL); and 3) decreasing cytokine (TNF-α and IL-1β) and chemokine (CCL2, CXCL10) levels in the retinal tissue. Inhibition of activated microglia with minocycline or the blockade of cytokines (TNF-α and IL-1β) with a receptor antagonist (RA) attenuated neuronal apoptosis after exposure to ketamine. ConclusionsThe upregulation of integrin β1 receptors in the microglia acts as a signaling molecule, triggering microgliosis to aggravate ketamine-induced neuronal apoptosis via the release of TNF-α and IL-1β in the early developing rat retina.

Genistein attenuates cognitive deficits and neuroapoptosis in hippocampus induced by ketamine exposure in neonatal rats.

Ketamine is a frequently used anesthetic in pediatric patients that can cause cognitive impairment. Genistein, a bioactive component of soy products, has been shown to suppress neuronal death through regulating the expression of apoptosis related genes. In this study, we hypothesized that genistein could alleviate ketamine-induced cognitive impairment by ameliorating hippocampal neuronal loss and tested this hypothesis in rats. Neonatal rats were treated with ketamine and genistein. Hippocampal tissue was harvested for histological and biochemical analysis to determine neuronal apoptosis and proteins involved in the apoptotic pathways. Behavioral assays including contextual fear conditioning test and Morris water maze test were performed to assess cognitive functions, including learning and memory. We found that in fear conditioning test, genistein restored freezing time in ketamine treated rats in a dose dependent manner. Similarly, genistein attenuated impaired learning and memory in Morris water maze test in rats treated with ketamine. Additionally, ketamine-induced neuronal apoptosis in rat hippocampus was attenuated by genistein treatment. Finally, we found that genistein partially restored proteins associated with apoptosis, including Bax, Bcl-2, cleaved caspase 3, and phosphorylated GSK-3ß and Akt. Genistein suppresses hippocampal neuronal loss and cognitive disruption induced by ketamine in rats.

Laminin degradation by matrix metalloproteinase 9 promotes ketamine-induced neuronal apoptosis in the early developing rat retina.

AimsDuring early development, laminin degradation contributes to the death of neurons. This study aims to investigate the role and regulation of laminin in ketamine‐induced apoptosis.MethodsWe performed terminal deoxynucleotidyl transferase biotin‐dUTP nick end labeling (TUNEL) and immunohistochemical assays to investigate the roles of the non‐integrin laminin receptor, matrix metalloproteinase 9 (MMP9) in ketamine‐induced neuronal apoptosis. In situ zymography, Western blot, and immunofluorescence were used to explore the relationships between laminin, MMP9 activity, and Zn2+. Experiments were performed using whole‐mount retinas dissected from Sprague Dawley rats.ResultsThe TUNEL and immunohistochemical assays indicated that ketamine‐induced neuronal apoptosis in early developing rat retina. Blockade of non‐integrin laminin receptor promoted ketamine‐induced apoptosis, while non‐integrin laminin receptor activation attenuated ketamine‐induced apoptosis. Ketamine‐induced laminin degradation, possibly by enhancing the activity of MMP9. MMP9 inhibition reduced ketamine‐induced apoptosis by reducing laminin degradation. Downregulation of Zn2+ attenuated the increased MMP9 activity, laminin degradation caused by ketamine and significantly reduced ketamine‐induced neuronal apoptosis.ConclusionLaminin degradation by MMP9 promoted ketamine‐induced neuronal apoptosis in early developing rat retina. The non‐integrin laminin receptor may be a pathway involved in ketamine‐induced apoptosis. Zn2+ downregulation may play a protective role against ketamine‐induced neuronal apoptosis through inhibiting MMP9 activity.

Traditional Chinese medicine, Kami-Shoyo-San protects ketamine-induced neurotoxicity in human embryonic stem cell-differentiated neurons through activation of brain-derived neurotrophic factor.

Anesthesia-induced neurotoxicity may cause permanent dysfunctions in human brains. In this work, we used a cell-based in-vitro model to demonstrate that traditional Chinese medicine, Kami-Shoyo-San may protect ketamine-induced neuronal apoptosis in human embryonic stem cell-differentiated neurons. Human embryonic stem cell-differentiated neurons were cultured in vitro and treated with high-concentration ketamine to induce neuronal apoptosis. Pre-incubation of Kami-Shoyo-San was conducted to evaluate its neuroprotection on ketamine-injured neurons. Quantitative real-time PCR and western blot assays were used to assess brain-derived neurotrophic factor and its receptor, tropomyosin receptor kinase B, in response to Kami-Shoyo-San and ketamine treatment. Brain-derived neurotrophic factor/tropomyosin receptor kinase B signaling pathway was then deactivated, by siRNA application, to further explore its functional role in Kami-Shoyo-San-mediated protection on ketamine-induced apoptosis among human embryonic stem cell-differentiated neurons. High concentration of ketamine-induced significant apoptosis, whereas pre-incubation of Kami-Shoyo-San markedly rescued ketamine-induced apoptosis, in human embryonic stem cell-differentiated neurons. Kami-Shoyo-San activated brain-derived neurotrophic factor/tropomyosin receptor kinase B signaling pathway by upregulating brain-derived neurotrophic factor and inducing tropomyosin receptor kinase B phosphorylation. Conversely, siRNA-mediated brain-derived neurotrophic factor/tropomyosin receptor kinase B signaling pathway deactivation reversed the neuroprotection of Kami-Shoyo-San in ketamine-injured human embryonic stem cell-differentiated neurons. Kami-Shoyo-San could protect ketamine-induced neurotoxicity, and the underlying mechanism may involve brain-derived neurotrophic factor/tropomyosin receptor kinase B signaling pathway.

Ketamine exacerbates cortical neuroapoptosis under hyperoxic conditions by upregulating expression of the N-methyl-D-aspartate receptor subunit NR1 in the developing rat brain

BackgroundKetamine and hyperoxia are widely used in obstetric and pediatric settings. Either ketamine or hyperoxia has been reported to cause neuroapoptosis in the developing brain, and ketamine-induced neuronal apoptosis may involve a compensatory upregulation of the N-methyl-D-aspartate (NMDA) receptor NR1 subunit. This study investigated the impact of ketamine administration under hyperoxic conditions on cortical neuroapoptosis and NR1 subunit expression in the infant rat brain.MethodsMale, 7-day-old rats were randomly allocated to four groups: control, ketamine, hyperoxia, and ketamine + hyperoxia (n = 18 per group). Rats in the control and ketamine groups received subcutaneous injections of either vehicle (saline) or ketamine (50 mg/kg) in room air (21% oxygen). The hyperoxia and ketamine + hyperoxia groups were exposed to 60% oxygen for 2 h after receiving saline or ketamine. Physiological parameters and arterial oxygen saturation were observed. Neuronal apoptosis and the expressions of NR1 mRNA and protein in the frontal cortex were also examined by transferase dUTP nick end labeling (TUNEL) assays, qPCR and Western blot, respectively.ResultsKetamine alone had no effect on paO2 (P > 0.05), but pups exposed to hyperoxia or hyperoxia + ketamine had significantly greater paO2 values compared to control animals (P < 0.01). Animals exposed to ketamine and ketamine + hyperoxia showed higher apoptotic scores, mRNA and protein expression levels of NR1 than control animals (P < 0.01), and ketamine + hyperoxia caused a significantly greater increase than ketamine alone (P < 0.01).ConclusionsThese data suggest that ketamine administration under hyperoxic conditions exacerbates cortical neuroapoptosis in the developing brain, which may be closely associated with an enhancement in NMDA receptor NR1 subunit expression.

Nicotinic Acetylcholine Receptors are Associated with Ketamine-induced Neuronal Apoptosis in the Developing Rat Retina

Early synchronized spontaneous network activity is a hallmark of the brain growth spurt period, during which general anesthetics cause widespread neuronal apoptosis and subsequent cognitive dysfunction. However, the relationship of such activity to anesthetic-induced neuronal apoptosis remains to be determined. In this study, we utilized patch-clamp electrophysiological recording, immunohistochemistry, and TUNEL assays to investigate the potential roles of spontaneous network activity in ketamine-induced neuronal apoptosis during early development. All experiments were performed using acutely dissected whole-mount Sprague–Dawley rat retinas (0–14 postnatal days [P0–P14]). Ketamine reversibly blocked spontaneous network activity in the rat retina from P0 to P9 and irreversibly blocked such activity from P10 to P12. The peak of physiological and ketamine-induced neuronal apoptosis mainly occurred from P7 to P9. Blockade of nicotinic acetylcholine receptors (nAChRs) also induced reversible inhibition of spontaneous network activity from P0 to P7 and extensive neuronal apoptosis in the P7 rat retina, while activation of nAChRs or increases in endogenous ACh levels attenuated ketamine-induced apoptotic responses. Furthermore, blockade of α7-nAChR and ß2-nAChR subtypes induced neuronal apoptosis in the developing retina, while activation of the α7-nAChR subtype attenuated ketamine-induced apoptotic responses. These results demonstrate that ketamine may inhibit early synchronized spontaneous network activity by blocking nAChRs, and that such inhibition may contribute to ketamine-induced neuronal apoptosis in the developing rat retina.

Ketamine induces neuronal apoptosis and cognitive disorder via miR-199a-5p/HIF-1α in neonatal rats

It reported that repeated administration of clinical doses of ketamine could induce neuronal apoptosis in the immature brain. In this study, Sprague-Dawley postnatal day 7 rats were treated with ketamine. As the rats grew, we found ketamine impaired the cognitive function of the rats. Ketamine increased HIF-1α level in the brain tissues of rats as compared to the control group and YC-1 rescued those effects. The escape latency and the platform crossing time of Morris water maze decreased in the YC-1-treated rats compared to the control in ketamine-treated rats. We also found that ketamine decreased the expression of miR-199a-5p and HIF-1α is a directly target of miR-199a-5p. Administration of ketamine also decrease the cell activity while YC-1 and MiR-199a-5p mimics rescued the inhibition effect. In conclusion, we suggest that ketamine-induced neuronal apoptosis in neonatal rats, followed by learning and memory impairment, might be mediated via the miR-199a-5p/HIF-1α signalling pathway.

Inhibition of long non-coding RNA IGF2AS protects apoptosis and neuronal loss in anesthetic-damaged mouse neural stem cell derived neurons.

In vitro culture of neural stem cell-derived neurons serves as an excellent model to study anesthetic-induced neurotoxicity. In our study, we examined the functional role of long non-coding RNA, IGF2AS, in regulating ketamine-induced neurotoxicity in murine neural stem cells. Murine E18.5 brain-derived neural stem cells were cultured in vitro. During neural differentiation stage, ketamine-induced gene expression changes of IGF2 and IGF2AS were recorded by qRT-PCR. Neural stem cell culture was then infected by IGF2AS siRNA. The protective effect of IGF2AS inhibition on ketamine-induced apoptosis and neurite loss was assessed by TUNEL and neurite growth assays. BDNF and Akt, two candidates of downstream signaling pathways associated with IGF2AS inhibition, were further examined by western blot in neural stem cell culture. In neural stem cell culture, IGF2 was downregulated, but IGF2AS upregulated by in vitro treatment of ketamine in dose-dependent manner. Transfection of IGF2AS-specific siRNA effectively downregulated endogenous IGF2AS expression in neural stem cells. In addition, IGF2AS inhibition significantly alleviated ketamine-induced neuronal apoptosis and neurite loss in neural stem cell-derived neurons. Western blot study revealed that IGF2AS inhibition upregulated downstream pro-neuronal signaling pathway proteins BDNF and Akt. Inhibiting endogenous IGF2AS can protect anesthetic-induced neurotoxicity in neural stem cells, possibly through complimentary IGF2 upregulation and its associated downstream signaling pathways.

Dopamine Attenuates Ketamine-Induced Neuronal Apoptosis in the Developing Rat Retina Independent of Early Synchronized Spontaneous Network Activity.

Deprivation of spontaneous rhythmic electrical activity in early development by anesthesia administration, among other interventions, induces neuronal apoptosis. However, it is unclear whether enhancement of neuronal electrical activity attenuates neuronal apoptosis in either normal development or after anesthesia exposure. The present study investigated the effects of dopamine, an enhancer of spontaneous rhythmic electrical activity, on ketamine-induced neuronal apoptosis in the developing rat retina. TUNEL and immunohistochemical assays indicated that ketamine time- and dose-dependently aggravated physiological and ketamine-induced apoptosis and inhibited early-synchronized spontaneous network activity. Dopamine administration reversed ketamine-induced neuronal apoptosis, but did not reverse the inhibitory effects of ketamine on early synchronized spontaneous network activity despite enhancing it in controls. Blockade of D1, D2, and A2A receptors and inhibition of cAMP/PKA signaling partially antagonized the protective effect of dopamine against ketamine-induced apoptosis. Together, these data indicate that dopamine attenuates ketamine-induced neuronal apoptosis in the developing rat retina by activating the D1, D2, and A2A receptors, and upregulating cAMP/PKA signaling, rather than through modulation of early synchronized spontaneous network activity.

Baicalin Attenuates Ketamine-Induced Neurotoxicity in the Developing Rats: Involvement of PI3K/Akt and CREB/BDNF/Bcl-2 Pathways.

Ketamine is widely used as an anesthetic in pediatric clinical practice. However, numerous studies have reported that exposure to ketamine during the developmental period induces neurotoxicity. Here we investigate the neuroprotective effects of baicalin, a natural flavonoid compound, against ketamine-induced apoptotic neurotoxicity in the cortex and hippocampus of the Sprague-Dawley postnatal day 7 (PND7) rat pups. Our results revealed that five continuous injections of ketamine (20mg/kg) at 90-min intervals over 6h induced obvious morphological damages of neuron by Nissl staining and apoptosis by TUNEL assays in the prefrontal cortex and hippocampus of PND7 rat pups. Baicalin (100mg/kg) pretreatment alleviated ketamine-induced morphological change and apoptosis. Caspase-3 activity and caspase-3 mRNA expression increase induced by ketamine were also inhibited by baicalin treatment. LY294002, an inhibitor of PI3K, abrogated the effect of baicalin against ketamine-induced caspase-3 activity and caspase-3 mRNA expression increase. In addition, Western blot studies indicated that baicalin not only inhibited ketamine-induced p-Akt and p-GSK-3β decrease, but also relieved ketamine-induced p-CREB and BDNF expression decrease. Baicalin also attenuated ketamine-induced Bcl-2/Bax decrease and caspase-3 expression increase. Further in vitro experiments proved that baicalin mitigated ketamine-induced cell viability decrease in the MTT assay, morphological change by Rosenfeld's staining, and caspase-3 expression increase by Western blot in the primary neuron-glia mixed cultures. LY294002 abrogated the protective effect of baicalin. These data demonstrate that baicalin exerts neuroprotective effect against ketamine-induced neuronal apoptosis by activating the PI3K/Akt and its downstream CREB/BDNF/Bcl-2 signaling pathways. Therefore, baicalin appears to be a promising agent in preventing or reversing ketamine's apoptotic neurotoxicity at an early developmental stage.

17β‐Oestradiol Protects Primary‐Cultured Rat Cortical Neurons from Ketamine‐Induced Apoptosis by Activating PI3K/Akt/Bcl‐2 Signalling

Numerous studies in rodents have indicated that exposure to ketamine during the period when synaptogenesis is highly active induces neurodegeneration. Thus, there is a growing need to develop strategies to prevent ketamine-induced brain injury in the developing brain. Oestradiol is a neuroactive steroid that prevents neuronal cell death in different experimental models by activating cell survival signals and inhibiting apoptotic signals. The main goal of this study was to investigate the neuroprotective effects of 17β-oestradiol against ketamine-induced apoptotic neurodegeneration in primary-cultured cortical neurons. The data revealed that 17β-oestradiol (0.1μM) in combination with ketamine (100μM) increased cell viability in the MTT assay and reduced the number of apoptotic cells detected by TUNEL and Hoechst 33258 staining. To elucidate a possible mechanism by which 17β-oestradiol exerts its neuroprotective effect, we investigated the PI3K pathway using an inhibitor of PI3K, LY294002. The protective effects of 17β-oestradiol were abrogated by LY294002. Furthermore, we found that 17β-oestradiol not only induced phosphorylation of the PI3K substrate Akt, but also increased the expression of Bcl-2, which down-regulated ketamine-induced caspase-3 activity and inhibited neuronal apoptosis. These data demonstrate that 17β-oestradiol exerts a neuroprotective effect against ketamine-induced neuronal apoptosis by activating the PI3K/Akt/Bcl-2 signalling pathway. Therefore, 17β-oestradiol appears to be a promising agent in preventing or reversing ketamine's toxic effects on neurons at an early developmental stage.

MicroPET imaging of ketamine-induced neuronal apoptosis with radiolabeled DFNSH

Recent reports indicate that 6-12 h of ketamine anesthesia can trigger neuronal apoptosis in postnatal day (PND) 7 rats. In vitro, ex vivo, and confocal fluorescent imaging studies suggest that dansyl compounds can accumulate within the cytoplasm of the apoptotic cell. High-resolution positron emission tomography (microPET) imaging has been proposed as a minimally invasive method for detecting apoptosis in the rat brain. Compared with [(18)F]-labeled annexin V, which binds to externalized phosphatidylserine (PS) on the outer membrane of apoptotic cells, intracellular uptake of the dansylhydrazone of p-fluorobenzaldehyde (DFNSH) may lead to improved target-to-background contrast ratios. In this study, the effect of ketamine on the uptake and retention of [(18)F]-DFNSH in the rat brain was investigated using microPET imaging. On PND 7, rat pups in the experimental group were exposed, at 2-h intervals, to six subcutaneous injections of ketamine (20 mg/kg) and control rat pups received six injections of saline. On PND 35, [(18)F]-DFNSH (37 MBq) was injected into the tail vein of rats and microPET images were obtained over 2 h following the injection. Radiolabeled tracer accumulation in the region of interest (ROI) in the frontal cortex was converted into standard uptake values (SUVs). The radiotracer was quickly distributed into the brains of both ketamine- and saline-treated rats. Compared with the control group, the uptake of [(18)F]-DFNSH was significantly increased in the ROI, frontal cortex area of ketamine-treated rats. In addition, the wash-out duration of the tracer was prolonged in the ketamine-treated animals. This study demonstrates that microPET imaging is capable of distinguishing differences in retention of [(18)F]-DFNSH in ROI and suggests that this compound may serve as a minimally invasive biomarker of neuronal apoptosis in rodents.

Effect of propofol on ketamine-induced cognitive dysfunction in aged rats

Objective To investigate the effect of propofol on ketamine-induced cognitive dysfunction and neuronal apoptosis in hippocampus in aged rats. Methods Thirty-two male SD rats aged 18-24 months weighing 380-470 g were randomly divided into 4 groups ( n = 8 each) :control group (group C);propofol group (group P);ketamine group (group K) and propofol + ketamine group (group PK). Propofol 30 mg·kg-1·h-1 or/and ketamine 40 mg· kg-1·h-1 were infused for 2 h once a day for 7 consecutive days. After the last day of drug administration cognitive function was assessed using Morris water maze (escape latency and the number of animals' swimming across the platform). The animals were sncrificed after water naze test and their hippocampi were removed for determination of neuronal apoptosis (by TUNEL) and caspase-3 expression (by immuno-histochemistry) in hippocampal CA1 region. Results There was no significant difference in escape latency and the number of the animals,swimming across the platform, the neuronal apoptotic rate (the number of apoptotic neurons/the number of total neurons) and caspase-3 expression between group C and P. In group K and PK the escape latency was prolonged,the number of animals' swimming across the platform was decreased, neuronal apoptotic rate increased and the caspase-3 expression up-regulated as compared with group C. The ketamine-induced changes were significantly attenuated by coadministration of propofol in group PK. Conclusion Coadministration of propofol can ameliorate ketamine-induced cognitive dysfunction and hippocampal neuronal apoptosis. Key words: Propofol; Ketamine; Cognition disorders; Aged