Buckwheat tartary regulates the Gsk-3ß/ß-catenin pathway to prevent neurobehavioral impairments in a rat model of surgical menopause.

Menopause is a natural aging process characterized by decreased levels of sex hormones in females. Deprivation of estrogen following menopause results in alterations of dendritic arborization of the neuron that leads to neurobehavioral complications. Hormone replacement therapy is in practice to manage postmenopausal conditions but is associated with a lot of adverse effects. In the present study, the efficacy of buckwheat tartary (Fagopyrum tataricum) whole seed extract was investigated against the neurobehavioral complication in middle-aged ovariectomized rats, which mimic the clinical postmenopausal condition. Hydroalcoholic extraction (80% ethanol) was done, and quantification of major marker compounds in the extract was performed using HPLC. Oral treatment of the extract following the critical window period rescued the reconsolidation process of spatial and recognition memory, as well as depression-like behavior. Gene expression analysis disclosed elevated oxidative stress and neuroinflammation that largely disturb the integrity of the blood-brain barrier in ovariectomized rats. Gfap and Pparγ expression also showed reactive astrogliosis in the rats subjected to ovariectomy. The extract treatment reverted the elevated oxidative stress, neuroinflammation and expression of the studied genes. Furthermore, protein expression analysis revealed that Gsk-3ß was activated differentially in the brain, as suggested by ß-catenin protein expression, which was normalized following the treatment with extract and rescued the altered neurobehavioral process. The results of the current study concluded that Fagopyrum tataricum seed extract is better option to overcome the neurobehavioral complications associated with the menopause.

Deciphering key regulators involved in epilepsy-induced cardiac damage through whole transcriptome and proteome analysis in a rat model.

OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) is a major outcome of cardiac dysfunction in patients with epilepsy. In continuation of our previous work, the present study was envisaged to explore the key regulators responsible for cardiac damage associated with chronic seizures using whole transcriptome and proteome analysis in a rat model of temporal lobe epilepsy. METHODS: A standard lithium-pilocarpine protocol was used to induce recurrent seizures in rats. The isolated rat heart tissue was subjected to transcriptomic and proteomic analysis. An integrated approach of RNA-Seq, proteomics, and system biology analysis was used to identify key regulators involved in seizure-linked cardiac changes. The analyzed differential expression patterns and network interactions were supported by gene and protein expression studies. RESULTS: Altogether, 1157 differentially expressed genes and 1264 proteins were identified in the cardiac tissue of epileptic animals through RNA-Seq and liquid chromatography with tandem mass spectrometry-based proteomic analysis, respectively. The network analysis revealed seven critical genes-STAT3, Myc, Fos, Erbb2, Erbb3, Notch1, and Mapk8-that could play a role in seizure-mediated cardiac changes. The LC-MS/MS analysis supported the activation of the transforming growth factor ß (TGF-ß) pathway in the heart of epileptic animals. Furthermore, our gene and protein expression studies established a key role of STAT3, Erbb, and Mapk8 to develop cardiac changes linked with recurrent seizures. SIGNIFICANCE: The present multi-omics study identified STAT3, Mapk8, and Erbb as key regulators involved in seizure-associated cardiac changes. It provided a deeper understanding of molecular, cellular, and network-level operations of the identified regulators that lead to cardiac changes in epilepsy.

ß-Hydroxybutyrate Improves the Redox Status, Cytokine Production and Phagocytic Potency of Glucose-Deprived HMC3 Human Microglia-like Cells.

Brain glucose deprivation is a component of the pathophysiology of ischemia, glucose transporter1 (GLUT1) deficiency, neurological disorders and occurs transiently in diabetes. Microglia, the neuroimmune cells must function effectively to offer immune defence and debris removal in low-energy settings. Brain glucose deprivation may compromise microglial functions further escalating the disease pathology and deteriorating the overall mental health. In the current study, HMC3 human microglia-like cells were cultured in vitro and exposed to glucose deprivation to investigate the effects of glucose deprivation on phenotypic state, redox status, secretion of cytokines and phagocytic capabilities of HMC3 cells. However, HMC3 cells were able to proliferate in the absence of glucose but showed signs of redox imbalance and mitochondrial dysfunction, as demonstrated by decreased MTT reduction and Mito Tracker™ staining of cells, along with a concomitant reduction in NOX2 protein, superoxide, and nitrite levels. Reduced levels of secreted TNF and IL-1ß were the signs of compromised cytokine secretion by glucose-deprived HMC3 microglia-like cells. Moreover, glucose-deprived HMC3 cells also showed reduced phagocytic activity as assessed by fluorescently labelled latex beads-based functional phagocytosis assay. ß-hydroxybutyrate (BHB) supplementation restored the redox status, mitochondrial health, cytokine secretion, and phagocytic activity of glucose-deprived HMC3 microglia-like cells. Overall, impaired brain glucose metabolism may hinder microglia's capacity to release diffusible immune factors and perform phagocytosis. This could escalate the mental health issues in neurological diseases where brain glucose metabolism is compromised. Moreover, nutritional ketosis or exogenous ketone supplementation such as BHB may be utilized as a potential metabolic therapies for these conditions.

Protective effect of Nardostachys jatamansi extract against lithium-pilocarpine-induced spontaneous recurrent seizures and associated cardiac irregularities in a rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Nardostachys jatamansi (D.Don) DC. is a perennial herbaceous medicinal plant widely used for the ethnomedical treatment of various ailments. The underground parts of the plants are used in traditional medicine to manage epilepsy and other cardiovascular conditions. AIM OF THE STUDY: The present study was undertaken to investigate the efficacy of a characterized hydroalcoholic extract (NJET) of Nardostachys jatamansi in the lithium-pilocarpine rat model of spontaneous recurrent seizures (SRS) and associated cardiac irregularities. MATERIALS AND METHODS: NJET was prepared by percolation using 80% ethanol. The dried NEJT was subjected to UHPLC-qTOF-MS/MS for chemical characterization. Molecular docking studies were performed using the characterized compounds to understand mTOR interactions. The animals showing SRS following lithium-pilocarpine administration were treated with NJET for 6 weeks. Afterward, seizure severity, cardiac parameters, serum biochemistry, and histopathological parameters were studied. The cardiac tissue was processed for specific protein and gene expression studies. RESULTS: The UHPLC-qTOF-MS/MS characterized 13 compounds in NJET. The identified compounds subjected to molecular docking showed promising binding affinities toward mTOR. There was a dose-dependent decrease in the severity of SRS following the extract administration. A reduction in mean arterial pressure and serum biochemical markers (lactate dehydrogenase and creatine kinase) was also observed following NJET treatment in epileptic animals. Histopathological investigations revealed reduced degenerative changes and decreased fibrosis following the extract treatment. The cardiac mRNA level of Mtor, Rps6, Hif1a, and Tgfb3 was reduced in the extract-treated groups. Further, a similar reduction in the protein expression of p-mTOR and HIF-1α was also observed following NJET treatment in the cardiac tissue. CONCLUSIONS: The results concluded that NJET treatment reduces lithium-pilocarpine-induced recurrent seizures and associated cardiac irregularities via downregulation of the mTOR signalling pathway.

Lithium co-administration with rutin improves post-stroke neurological outcomes via suppressing Gsk-3ß activity in a rat model.

Cerebral ischemic stroke is one of the leading causes of adult disability worldwide. Reperfusion is the only therapeutic option with a lot of side effects. In the current study, we investigated the efficacy of rutin and lithium co-treatment in improving post-stroke neurological outcomes in a transient global cerebral ischemia-reperfusion injury rat model. Middle-aged male rats were subjected to transient global cerebral ischemia-reperfusion. NORT and Y-maze were used to assess the cognitive processes. Lipid peroxidation, protein carbonylation, and nitric oxide assays were performed to study oxidative stress. The excitotoxicity index was calculated by HPLC. Real time-PCR and western blotting were performed to study gene and protein expressions. The co-administration of rutin and lithium improved the overall survival, recognition memory, spatial working memory, and neurological score following cerebral ischemia-reperfusion in rats. Further, a marked decrease in malonaldehyde, protein carbonyls, and nitric oxide levels was observed following combined treatment. The mRNA expression of antioxidant (Hmox1 and Nqo1) and pro-inflammatory (Il2, Il6, and Il1ß) markers were significantly attenuated in the rutin and lithium co-administrated group. The treatment inhibited the Gsk-3ß and maintained a normal pool of the downstream ß-catenin and Nrf2 proteins. The results revealed that co-administration of rutin and lithium had a neuroprotective potential, suggesting it to be a viable treatment to overcome post-stroke deaths and neurological complications.

Inhibition of Mammalian Target of Rapamycin Attenuates Recurrent Seizures Associated Cardiac Damage in a Zebrafish Kindling Model of Chronic Epilepsy.

Sudden Unexpected Death in Epilepsy (SUDEP) is primarily linked with the cardiac irregularities that occur due to recurrent seizures. Our previous studies found a role of mTOR pathway activation in seizures-linked cardiac damage in a rat model. In continuation to the earlier work, the present study was devised to explore the role of rapamycin (mTOR inhibitor and clinically used immunosuppressive agent) in a zebrafish kindling model and associated cardiac damage. Adult zebrafish were incubated with increasing concentrations of rapamycin (1, 2 and, 4 µM), followed by pentylenetetrazole (PTZ) exposure to record seizure latency and severity. In another experiment, zebrafish were subjected to a standardized PTZ kindling protocol. The kindled fish were treated daily with rapamycin for up to 25 days, along with PTZ to record seizure severity. At the end, zebrafish heart was excised for carbonylation assay, gene expression, and protein quantification studies. In the acute PTZ convulsion test, treatment with rapamycin showed a significant increase in seizure latency and decreased seizure severity without any change in seizure incidence. Treatment with rapamycin also reduced the severity of seizures in kindled fish. The cardiac expressions of gpx, nppb, kcnh2, scn5a, mapk8, stat3, rps6 and ddit were decreased, whereas the levels of trxr2 and beclin 1 were increased following rapamycin treatment in kindled fish. Furthermore, rapamycin treatment also decreased p-mTOR expression and protein carbonyls level in the fish cardiac tissue. The present study concluded that rapamycin reduces seizures and associated cardiac damage by inhibiting mTOR activation in the zebrafish kindling model.

Lithium therapy subdues neuroinflammation to maintain pyramidal cells arborization and rescues neurobehavioural impairments in ovariectomized rats.

Oestrogen deprivation as a consequence of menopause alters the brain neuronal circuit and results in the development of neurobehavioural symptoms later. Hormone replacement therapy to some extent helps to overcome these abnormalities but is associated with various adverse events. Lithium therapy is being used to manage multiple neuropsychiatric disorders and is reported to maintain structural synaptic plasticity, suppress neuroinflammation, and promote adult neurogenesis. The present study examined the effect of lithium treatment on the neurobehavioural impairments in ovariectomized rat model mimicking clinical postmenopausal condition. A protective effect of lithium treatment was observed on the reconsolidation of spatial and recognition memory along with depression-like behaviour in ovariectomized rats. The Golgi-Cox staining revealed increased dendritic length and spine density in the pyramidal neurons of the CA1 region of the hippocampus, layer V of the somatosensory cortex, and layer II/III of the prefrontal cortex in the treated group. A significant reduction in pro-inflammatory markers, Il2, II6, and Il1b, was observed in the hippocampus, somatosensory cortex, and prefrontal cortex following lithium treatment. mRNA expression studies of Gfap and Pparg, along with histopathological analysis, suggested reactive astrogliosis to be a major contributor of neuroinflammation in ovariectomized rats that was normalized following lithium treatment. Further, the treatment inhibited Gsk-3ß activity and maintained the normal level of ß-catenin, CREB, and BDNF. The results revealed a defensive role of lithium against ovariectomy-induced neurobehavioural impairments, thus suggesting it to be a potential therapeutic agent for managing postmenopausal neurological symptoms.

Rutin protects hemorrhagic stroke development via supressing oxidative stress and inflammatory events in a zebrafish model.

Intracerebral hemorrhagic (ICH) stroke is a major cause of death and disability globally, with no proper treatment available so far. Rutin, a dietary flavonoid, has shown protection against cerebral ischemic stroke due to its antioxidant and anti-inflammatory attributes. However, the efficacy of rutin against ICH stroke remained unexplored. Therefore, in the current study, we investigated the effect of rutin in an ICH stroke zebrafish larva model. The larvae were exposed to atorvastatin (1.25  µM) in system water for induction of experimental ICH. Rutin treatment reduced the hematoma size, ROS production and decreased apoptosis in the zebrafish larvae brains. Reduction in the malondialdehyde and protein carbonyl level in the rutin-treated larvae also indicated quenching of the free radicals. The treatment increased the expression of tight junction claud5a gene and decreased the mRNA level of matrix metalloproteases (mmp2 and mmp9). Furthermore, rutin treatment also attenuated the genomic expression of oxidative markers (nrf2, hmox1a, sod1, and gpx) and inflammatory genes (il6, tnfa, il10, and irf2a) related to ICH. The Gsk-3ß activity was also downregulated, and a normal pool of ß-catenin and Nrf2 was maintained in the larvae treated with rutin. The current study suggested that rutin protects ICH stroke via suppressing oxidative stress and inflammatory events in a zebrafish model.

Recognition of Acute Page Kidney following Renal Transplant: Varied Etiologies Requiring High Clinical Suspicion.

Oliguria in the early postoperative phase after renal transplantation has many causes with overlapping presentations. Page kidney refers to external compression of the kidney by a hematoma, urinoma or tumor, leading to parenchymal hypoperfusion, unexplained hypertension (HTN), or frank acute renal failure. About 100 cases of Page kidney are reported; mostly after kidney biopsy. After the analysis of records, we identified four cases of acute Page kidney posttransplant, akin to a compartment syndrome. All biochemical, laboratory, and clinical parameters were recorded. Cases occurred within two to three weeks of transplant, with different causes. Clinical presentation was sudden, with HTN, raised serum creatinine and perigraft swelling in all. Rejection co-existed Page kidney in two cases, while tacrolimus had to be potentiated with diltiazem in one case. Serial parameters such as increased resistive index (>0.7), perigraft collection, and absent diastolic flow with normal peak systolic velocity were consistent with diagnosis. Two were caused by lymphoceles, more than 3 L. Both were managed by laparoscopic fenestration surgery; probably the first such instance for Page kidney. Two patients had postoperative hematoma; in one case, it followed early percutaneous angiographic stenting and "leakage" from the transplant artery, only the second such report. A high index of suspicion required for diagnosis; after excluding rejection and pre/postrenal causes, aggressive early management is the key for graft salvage.

Can renal transplant improve the quality of life of caregiver donors? A prospective study from India.

Live-related renal transplantation in India by "caregiver donors" provides huge financial, emotional, and physical support. Their psychological and mental health has not been addressed. We performed a prospective study using the World Health Organization Quality of Life (WHOQoL) BREF Scores and the Hospital Anxiety and Depression Scales preoperatively, at two weeks and three months after transplant. We included 30 pairs; most donors were females (80%, 60% mothers, 28% wives). The mean age of donors was 43.77 ± 10.64 years (34.8 ± 9.01 for recipients). There was improvement in the WHOQoL BREF after two weeks and three months as follows: physical domain (74.30 ± 9.74 vs. 78.30 ± 8.20; P = 0.001), and (74.30 ± 9.74 vs. 86.23 ± 7.25; P <0.001); psychological (74.90 ± 8.44 vs. 82.07 ± 7.19; P <0.001) and (74.90 ± 8.44 vs. 88.07 ± 6.89; P <0.001); environmental (75.33 ± 8.09 vs. 79.57 ± 6.18; P <0.001), and, (75.33 ± 8.09 vs. 86.97 ± 3.8; P <0.001); social-relationships (77.73 ± 8.28 vs. 79.77 ± 7.99; P <0.001), and (77.73 ± 8.28 vs. 84.77 ± 7.45; P <0.001). The recipient scores were similar. Factors with significant Pearson's or standardized beta co-efficient were donor age <20 years, donor complications, donor anxiety, education (<12th standard), recipient hospital stay (>3 weeks), and, recipient complications (increased creatinine, hemodialysis, lymphocele, and graft dysfunction). The median anxiety scores of donors increased significantly two weeks after operation but later became normal. Caregiver donors have improved QoL scores, despite kidney donation; a larger study is needed.

Glycogen synthase kinase-3: A putative target to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic.

The coronavirus disease 19 (COVID-19) outbreak caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) had turned out to be highly pathogenic and transmittable. Researchers throughout the globe are still struggling to understand this strain's aggressiveness in search of putative therapies for its control. Crosstalk between oxidative stress and systemic inflammation seems to support the progression of the infection. Glycogen synthase kinase-3 (Gsk-3) is a conserved serine/threonine kinase that mainly participates in cell proliferation, development, stress, and inflammation in humans. Nucleocapsid protein of SARS-CoV-2 is an important structural protein responsible for viral replication and interferes with the host defence mechanism by the help of Gsk-3 protein. The viral infected cells show activated Gsk-3 protein that degrades the Nuclear factor erythroid 2-related factor (Nrf2) protein, resulting in excessive oxidative stress. Activated Gsk-3 also modulates CREB-DNA activity, phosphorylates NF-​κB, and degrades ß-catenin, thus provokes systemic inflammation. Interaction between these two pathophysiological events, oxidative stress, and inflammation enhance mucous secretion, coagulation cascade, and hypoxia, which ultimately leads to multiple organs failure, resulting in the death of the infected patient. The present review aims to highlight the pathogenic role of Gsk-3 in viral replication, initiation of oxidative stress, and inflammation during SARS-CoV-2 infection. The review also summarizes the potential Gsk-3 pathway modulators as putative therapeutic interventions in combating the COVID-19 pandemic.

Differential activation of Gsk-3ß in the cortex and the hippocampus induces cognitive and behavioural impairments in middle-aged ovariectomized rat.

Glycogen synthase kinase-3 (Gsk-3ß) aberration act as a crucial pathogenic factor in several neurological conditions. However its role in menopause associated behavioural impairments is still not unclear. The present study was designed to understand the role of Gsk-3ß in the progression of neurobehavioural impairments in middle-aged ovariectomized (ovx) rats. The animals showed a significant impairment in spatial and recognition memory, along with anxiety and depression-like behaviour following 22 weeks of ovx. The genomic expression of ERα, ERß, Nrf2, HO-1, TNFα, and IL-6 was altered in both the cortex and the hippocampus of ovx rats. Protein expression of p-Gsk-3ß(Ser9) was significantly downregulated in the cortex after ovx. However, the hippocampus showed a surprisingly opposite trend in the levels of p-Gsk-3ß(Ser9) as that of the cortex. Differential activation of Gsk-3ß and its downstream proteins such as ß-catenin and p-mTOR were also altered following ovx. The study concluded that differential activation of Gsk-3ß, along with oxidative stress and neuroinflammation in the cortex and the hippocampus, leads to the induction of cognitive and behaviour impairments in ovx rats.

Development and validation of chemical kindling in adult zebrafish: A simple and improved chronic model for screening of antiepileptic agents.

BACKGROUND: Zebrafish has emerged as a potential animal model of acute convulsion for early screening of antiepileptic agents. There is a need for alternative chronic zebrafish models of epilepsy with more correlation to the clinical condition. NEW METHOD: Adult zebrafish were repeatedly exposed to subeffective concentrations of pentylenetetrazole (PTZ), until appearance to tonic-clonic seizures, considered as kindled. Valproic acid (VPA) exposure was given during kindling and in kindled fish in 2 different groups. The neurotransmitters level and expression of the genes associated with kindling were studied in the fish brain. RESULTS: There was an increase in seizure severity score at 1.25 mM concentration of PTZ, and 66.66 % of fish achieved kindling after 22 days' exposure. A marked increase in c-fos, crebbpa and crebbpbexpression, and glutamate/GABA level was observed in the brain of kindled fish. VPA inhibited the induction of PTZ-mediated kindling and reduced seizure severity in kindled fish. COMPARISON WITH EXISTING METHOD: In contrast to an existing adult zebrafish kindling method, the present protocol is of longer duration, with more similarity to clinical epilepsy. Moreover, the induction of kindling involves a simple non-invasive technique without the use of anesthesia. The protocol can be used for evaluation of both antiepileptic and antiepileptogenic agents. CONCLUSION: Repeated exposure of 1.25 mM PTZ induced kindling in zebrafish, altering the brain neurotransmitter levels and gene expression. Inhibition of kindling induction and decrease in seizures in normal and kindled fish, respectively by VPA validated application of the model for preclinical testing of agents against epilepsy.

Targeting glycogen synthase kinase-3 for oxidative stress and neuroinflammation: Opportunities, challenges and future directions for cerebral stroke management.

Cerebral stroke is a leading cause of early death and physical disability in adults throughout the world. Oxidative stress and inflammation plays an important role in the pathological process associated with the stroke. The available reperfusion therapy itself enhances the oxidative stress by overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) from the neurons, as well as non-neuronal cells, thus further worsens the condition. Excessive ROS and RNS production contributes to the brain injury during or after the stroke by activating inflammatory processes. Glycogen synthase kinase-3 (Gsk-3) is an evolutionary conserved serine-threonine kinase that plays an important role in cellular growth, development, inflammation and apoptosis processes. Gsk-3 is activated during the brain stroke and suppresses the expression of nuclear factor-E2-related factor 2, an important regulator of endogenous antioxidant defence mechanism. It also modulates the expression of anti-inflammatory protein like, cAMP response element binding protein and activator protein 1. It has been found that the inhibition of Gsk-3 shows neuroprotection via reducing the oxidative stress and inflammation in cerebral ischemia/reperfusion. The present review describes in depth the role of oxidative stress, neuroinflammatory processes and endogenous defence systems involved in cerebral stroke pathogenesis, and their regulation by Gsk-3. Further the reports of all the potential agents modulating Gsk-3 activity via different signalling pathways have been summarized to substantiate its effectiveness in cerebral stroke management.

Approach to rectal foreign body: an unusual presentation.

Foreign bodies in the rectum are commonly confronted worldwide in the surgical emergency. Such a situation arises either accidentally or for autoerotic purposes. A wide variety of foreign objects have been reported in the literature and this usual object would add to the reports for its unusual location. We report a case of a 26-year-old young man with accidental insertion of hand shower in the rectum and no signs of any active bleed or peritonism. Extraction was done successfully in the operating room under general anaesthesia, with an uneventful postprocedure period and the patient was discharged after 48 hours of observation. Rectal foreign bodies can be of wide variety and can lead to life-threatening conditions. An orderly approach is required for diagnosis, management and evaluation in the postprocedure period.

Successful Transplant of Two Kidneys Harvested from a Young Brain-Dead Liver Transplant Recipient.

Efforts to increase the dismal deceased renal transplantation (DRT): live renal transplantation (LRT) ratio in our country have gathered momentum recently, with governmental and non-governmental projects focussing on building public awareness and capacity-building, and appropriate legislation. Worldwide, efforts at increasing the number of organs from the deceased pool have focussed on the use of 'expanded criteria donors', including deceased cardiac donors (DCD). 'Reuse' transplant, where an organ is transplanted after removal from the first recipient, is a rare strategy, used more commonly in liver than in kidney transplantation. Exceptional circumstances, where other organs have been harvested from transplant recipients, are rare. We describe the successful transplants of two renal grafts obtained from a 19-year-old brain-dead liver transplant recipient; this is probably the second case in English-language literature. A 19-year-old male patient with hepatitis E-induced fulminant hepatic failure underwent live-related liver transplantation. On postoperative day 2, cerebral edema set in, and the patient was declared brain-dead. Despite the economical and emotional trauma, the family opted for donation of the well-perfused kidneys. The kidneys were transported in HTK solution (histidine-tryptophan-ketoglutarate) to our centre. Recipient 1 was a 32-year-old woman (B positive) and recipient 2 was a 29-year-old man (also B positive); the kidneys were placed extraperitoneally and anastomosed end-to-side to the external iliac artery and vein. Recipient 2 experienced delayed graft function; however, both are doing well 15 months posttransplant.

Double gallbladder-a laparoscopic management.

A rare case of gallbladder duplication, an unusual biliary anomaly is reported in a young female patient presenting with acute cholecystitis. After a confirmed diagnosis of double gallbladder was made by sonography, endoscopic retrograde cholangio-pancreaticography (ERCP), and magnetic retrograde cholangio-pancreaticography(MRCP), both gallbladders were removed laparoscopically. On histology both gallbladders showed cholesterolosis. Detailed preoperative investigations are required for an accurate preoperative diagnosis before considering laparoscopic cholecystectomy to avoid inadvertent damage to biliary ductal system and overlooking of second gallbladder during surgery.