Neuroprotective role of Tinospora cordifolia extract in streptozotocin induced neuropathic pain

Abstract Diabetic Neuropathy (DN) is one of the prevailing micro vascular complications of diabetes which can be characterized by neuropathic pain. Streptozotocin (STZ) induced diabetes in the rat has been increasingly used as a model of painful diabetic neuropathy. STZ injection leads to neurotoxicity of peripheral nerves that leads to development of Peripheral Diabetic Neuropathy in rat model. The present study was aimed at exploring the protective role of Tinospora cordifolia extract in STZ induced neurotoxicity and evaluating mechanisms responsible for attenuating neuropathic pain. Neuropathic pain markers like hyperalgesia, allodynia and motor deficits were assessed before STZ injection and after the treatment with 250 mg/kg and 500 mg/kg dose of Tinospora cordifolia. Oxidative stress markers, NGF expression in sciatic nerve were observed after seven weeks treatment. Our results demonstrated that seven weeks treatment with Tinospora cordifolia leaf extract significantly relieved thermal hyperalgesia and allodynia by increasing the antioxidant enzyme levels, decreasing the lipid peroxidation and by increasing the Nerve growth factor (NGF) expression in diabetic rat sciatic nerves. Our findings highlighted the beneficial effects of oral administration of Tinospora cordifolia extract in attenuating diabetic neuropathic pain, possibly through a strong antioxidant activity and by inducing NGF m RNA in sciatic nerves.

Diet and companionship modulate pain via a serotonergic mechanism.

Treatment of severe chronic and acute pain in sickle cell disease (SCD) remains challenging due to the interdependence of pain and psychosocial modulation. We examined whether modulation of the descending pain pathway through an enriched diet and companionship could alleviate pain in transgenic sickle mice. Mechanical and thermal hyperalgesia were reduced significantly with enriched diet and/or companionship. Upon withdrawal of both conditions, analgesic effects observed prior to withdrawal were diminished. Serotonin (5-hydroxytryptamine, 5-HT) was found to be increased in the spinal cords of mice provided both treatments. Additionally, 5-HT production improved at the rostral ventromedial medulla and 5-HT accumulated at the dorsal horn of the spinal cord of sickle mice, suggesting the involvement of the descending pain pathway in the analgesic response. Modulation of 5-HT and its effect on hyperalgesia was also investigated through pharmaceutical approaches. Duloxetine, a serotonin-norepinephrine reuptake inhibitor, showed a similar anti-nociceptive effect as the combination of diet and companionship. Depletion of 5-HT through p-chlorophenylalanine attenuated the anti-hyperalgesic effect of enriched diet and companionship. More significantly, improved diet and companionship enhanced the efficacy of a sub-optimal dose of morphine for analgesia in sickle mice. These findings offer the potential to reduce opioid use without pharmacological interventions to develop effective pain management strategies.

Ketogenic diet effects on inflammatory allodynia and ongoing pain in rodents.

Ketogenic diets are very low carbohydrate, high fat, moderate protein diets used to treat medication-resistant epilepsy. Growing evidence suggests that one of the ketogenic diet's main mechanisms of action is reducing inflammation. Here, we examined the diet's effects on experimental inflammatory pain in rodent models. Young adult rats and mice were placed on the ketogenic diet or maintained on control diet. After 3-4 weeks on their respective diets, complete Freund's adjuvant (CFA) was injected in one hindpaw to induce inflammation; the contralateral paw was used as the control. Tactile sensitivity (von Frey) and indicators of spontaneous pain were quantified before and after CFA injection. Ketogenic diet treatment significantly reduced tactile allodynia in both rats and mice, though with a species-specific time course. There was a strong trend to reduced spontaneous pain in rats but not mice. These data suggest that ketogenic diets or other ketogenic treatments might be useful treatments for conditions involving inflammatory pain.

The analgesic effect of refeeding on acute and chronic inflammatory pain.

Pain is susceptible to various cognitive factors. Suppression of pain by hunger is well known, but the effect of food intake after fasting (i.e. refeeding) on pain remains unknown. In the present study, we examined whether inflammatory pain behavior is affected by 24 h fasting and 2 h refeeding. In formalin-induced acute inflammatory pain model, fasting suppressed pain behavior only in the second phase and the analgesic effect was also observed after refeeding. Furthermore, in Complete Freund's adjuvant-induced chronic inflammatory pain model, both fasting and refeeding reduced spontaneous pain response. Refeeding with non-calorie agar produced an analgesic effect. Besides, intraperitoneal (i.p.) administration of glucose after fasting, which mimics calorie recovery following refeeding, induced analgesic effect. Administration of opioid receptor antagonist (naloxone, i.p.) and cannabinoid receptor antagonist (SR 141716, i.p.) reversed fasting-induced analgesia, but did not affect refeeding-induced analgesia in acute inflammatory pain model. Taken together, our results show that refeeding produce analgesia in inflammatory pain condition, which is associated with eating behavior and calorie recovery effect.

A ketogenic diet reduces metabolic syndrome-induced allodynia and promotes peripheral nerve growth in mice.

Current experiments investigated whether a ketogenic diet impacts neuropathy associated with obesity and prediabetes. Mice challenged with a ketogenic diet were compared to mice fed a high-fat diet or a high-fat diet plus exercise. Additionally, an intervention switching to a ketogenic diet following 8 weeks of high-fat diet was performed to compare how a control diet, exercise, or a ketogenic diet affects metabolic syndrome-induced neural complications. When challenged with a ketogenic diet, mice had reduced bodyweight and fat mass compared to high-fat-fed mice, and were similar to exercised, high-fat-fed mice. High-fat-fed, exercised and ketogenic-fed mice had mildly elevated blood glucose; conversely, ketogenic diet-fed mice were unique in having reduced serum insulin levels. Ketogenic diet-fed mice never developed mechanical allodynia contrary to mice fed a high-fat diet. Ketogenic diet fed mice also had increased epidermal axon density compared all other groups. When a ketogenic diet was used as an intervention, a ketogenic diet was unable to reverse high-fat fed-induced metabolic changes but was able to significantly reverse a high-fat diet-induced mechanical allodynia. As an intervention, a ketogenic diet also increased epidermal axon density. In vitro studies revealed increased neurite outgrowth in sensory neurons from mice fed a ketogenic diet and in neurons from normal diet-fed mice given ketone bodies in the culture medium. These results suggest a ketogenic diet can prevent certain complications of prediabetes and provides significant benefits to peripheral axons and sensory dysfunction.

Effect of omega-3 polyunsaturated fatty acid treatment over mechanical allodynia and depressive-like behavior associated with experimental diabetes.

Neuropathic pain and depression are very common comorbidities in diabetic patients. As the pathophysiological mechanisms are very complex and multifactorial, current treatments are only symptomatic and often worsen the glucose control. Thus, the search for more effective treatments are extremely urgent. In this way, we aimed to investigate the effect of chronic treatment with fish oil (FO), a source of omega-3 polyunsaturated fatty acid, over the mechanical allodynia and in depressive-like behaviors in streptozotocin-diabetic rats. It was observed that the diabetic (DBT) animals, when compared to normoglycemic (NGL) animals, developed a significant mechanical allodynia since the second week after diabetes induction, peaking at fourth week which is completely prevented by FO treatment (0.5, 1 or 3g/kg). Moreover, DBT animals showed an increase of immobility frequency and a decrease of swimming and climbing frequencies in modified forced swimming test (MFST) since the second week after diabetes injection, lasting up at the 4th week. FO treatment (only at a dose of 3g/kg) significantly decreased the immobility frequency and increased the swimming frequency, but did not induce significant changes in the climbing frequency in DBT rats. Moreover, it was observed that DBT animals had significantly lower levels of BDNF in both hippocampus and pre frontal cortex when compared to NGL rats, which is completely prevented by FO treatment. In conclusion, our study demonstrates that FO treatment was able to prevent the mechanical allodynia and the depressive-like behaviors in DBT rats, which seems to be related to its capacity of BDNF level restoration.

Dietary fish oil inhibits mechanical allodynia and thermal hyperalgesia in diabetic rats by blocking nuclear factor-κB-mediated inflammatory pathways.

One of the most common complications of early-onset diabetes mellitus is peripheral diabetic neuropathy, which is manifested either by loss of nociception or by allodynia and hyperalgesia. Diabetes mellitus is a common metabolic disease in human beings with characteristic symptoms of hyperglycemia, chronic inflammation and insulin resistance. Dietary fatty acids, especially polyunsaturated fatty acids, have been shown anti-inflammatory role in various experimental conditions. The present study investigated the effects of fish oil supplementation on the inflammation in the dorsal root ganglion (DRG) of streptozotocin (STZ)-induced diabetes rats. The effects of diabetes and fish oil treatment on the allodynia and hyperalgesia were also evaluated. Dietary fish oil effectively attenuated both allodynia and hyperalgesia induce by STZ injection. Along with the behavioral findings, DRG from fish oil-treated diabetic rats displayed a decrease in inflammatory cytokines and the expression of nuclear factor-κB (NF-κB) compared with untreated diabetic rats. Fish oil supplementation also increased the phosphorylation of AKT in DRG of diabetic rats. These results suggested that dietary fish oil-inhibited allodynia and hyperalgesia in diabetic rats may stem from its anti-inflammatory potential by regulating NF-κB and AKT. Fish oil might be useful as an adjuvant therapy for the prevention and treatment of diabetic complications.

Effect of enriching the diet with menhaden oil or daily treatment with resolvin D1 on neuropathy in a mouse model of type 2 diabetes.

The purpose of this study was to determine the effect of supplementing the diet of a mouse model of type 2 diabetes with menhaden (fish) oil or daily treatment with resolvin D1 on diabetic neuropathy. The end points evaluated included motor and sensory nerve conduction velocity, thermal sensitivity, innervation of sensory nerves in the cornea and skin, and the retinal ganglion cell complex thickness. Menhaden oil is a natural source for n-3 polyunsaturated fatty acids, which have been shown to have beneficial effects in other diseases. Resolvin D1 is a metabolite of docosahexaenoic acid and is known to have anti-inflammatory and neuroprotective properties. To model type 2 diabetes, mice were fed a high-fat diet for 8 wk followed by a low dosage of streptozotocin. After 8 wk of hyperglycemia, mice in experimental groups were treated for 6 wk with menhaden oil in the diet or daily injections of 1 ng/g body wt resolvin D1. Our findings show that menhaden oil or resolvin D1 did not improve elevated blood glucose, HbA1C, or glucose utilization. Untreated diabetic mice were thermal hypoalgesic, had reduced motor and sensory nerve conduction velocities, had decreased innervation of the cornea and skin, and had thinner retinal ganglion cell complex. These end points were significantly improved with menhaden oil or resolvin D1 treatment. Exogenously, resolvin D1 stimulated neurite outgrowth from primary cultures of dorsal root ganglion neurons from normal mice. These studies suggest that n-3 polyunsaturated fatty acids derived from fish oil could be an effective treatment for diabetic neuropathy.

Commiphora mukul attenuates peripheral neuropathic pain induced by chronic constriction injury of sciatic nerve in rats.

OBJECTIVE: The management of neuropathic pain remains unsatisfactory till date, despite immense advances in the therapeutic strategies. Commiphora mukul (CM), also known as Commiphora wightii, is well known in the traditional Indian system of medicine, and has been used to treat ailments such as obesity, bone fractures, arthritis, inflammation, cardiovascular diseases, and lipid disorders. The present study was performed to investigate the effect of CM on peripheral neuropathic pain in rats. METHODS: Neuropathic pain was induced by the chronic constriction injury of the sciatic nerve. Following this, CM was orally administered for 2 weeks in doses of 50, 100, and 200 mg/kg, and pain assessment was performed by employing the behavioral tests for thermal hyperalgesia (hot-plate and tail-flick tests) and cold allodynia (acetone test). RESULTS: Following the induction of neuropathic pain, significant development of thermal hyperalgesia and cold allodynia was observed. The administration of CM (50 mg/kg) did not have any effect on the hot-plate and tail-flick tests, but significant anti-allodynic effect was observed in the acetone test. Furthermore, administration of CM (100 mg/kg) caused significant decrease in pain as observed on the tail-flick and acetone tests, but not in the hot-plate test. CM in a dose of 200 mg/kg significantly modulated neuropathic pain as observed from the increased hot-plate and tail-flick latencies, and decreased paw withdrawal duration (in acetone test). DISCUSSION: Therefore, the present study suggests that CM may be used in future as a treatment option for neuropathic pain.

Effects of methyl donor diets on incisional pain in mice.

BACKGROUND: Dietary supplementation with methyl donors can influence the programming of epigenetic patterns resulting in persistent alterations in disease susceptibility and behavior. However, the dietary effects of methyl donors on pain have not been explored. In this study, we evaluated the effects of dietary methyl donor content on pain responses in mice. METHODS: Male and female C57BL/6J mice were treated with high or low methyl donor diets either in the perinatal period or after weaning. Mechanical and thermal nociceptive sensitivity were measured before and after incision. RESULTS: Mice fed high or low methyl donor diets displayed equal weight gain over the course of the experiments. When exposed to these dietary manipulations in the perinatal period, only male offspring of dams fed a high methyl donor diet displayed increased mechanical allodynia. Hindpaw incision in these animals caused enhanced nociceptive sensitization, but dietary history did not affect the duration of sensitization. For mice exposed to high or low methyl donor diets after weaning, no significant differences were observed in mechanical or thermal nociceptive sensitivity either at baseline or in response to hindpaw incision. CONCLUSIONS: Perinatal dietary factors such as methyl donor content may impact pain experiences in later life. These effects, however, may be specific to sex and pain modality.

Metabolomics uncovers dietary omega-3 fatty acid-derived metabolites implicated in anti-nociceptive responses after experimental spinal cord injury.

Chronic neuropathic pain is a frequent comorbidity following spinal cord injury (SCI) and often fails to respond to conventional pain management strategies. Preventive administration of docosahexaenoic acid (DHA) or the consumption of a diet rich in omega-3 polyunsaturated fatty acids (O3PUFAs) confers potent prophylaxis against SCI and improves functional recovery. The present study examines whether this novel dietary strategy provides significant antinociceptive benefits in rats experiencing SCI-induced pain. Rats were fed control chow or chow enriched with O3PUFAs for 8weeks before being subjected to sham or cord contusion surgeries, continuing the same diets after surgery for another 8 more weeks. The paw sensitivity to noxious heat was quantified for at least 8weeks post-SCI using the Hargreaves test. We found that SCI rats consuming the preventive O3PUFA-enriched diet exhibited a significant reduction in thermal hyperalgesia compared to those consuming the normal diet. Functional neurometabolomic profiling revealed a distinctive deregulation in the metabolism of endocannabinoids (eCB) and related N-acyl ethanolamines (NAEs) at 8weeks post-SCI. We found that O3PUFAs consumption led to a robust accumulation of novel NAE precursors, including the glycerophospho-containing docosahexaenoyl ethanolamine (DHEA), docosapentaenoyl ethanolamine (DPEA), and eicosapentaenoyl ethanolamine (EPEA). The tissue levels of these metabolites were significantly correlated with the antihyperalgesic phenotype. In addition, rats consuming the O3PUFA-rich diet showed reduced sprouting of nociceptive fibers containing CGRP and dorsal horn neuron p38 mitogen-activated protein kinase (MAPK) expression, well-established biomarkers of pain. The spinal cord levels of inositols were positively correlated with thermal hyperalgesia, supporting their role as biomarkers of chronic neuropathic pain. Notably, the O3PUFA-rich dietary intervention reduced the levels of these metabolites. Collectively, these results demonstrate the prophylactic value of dietary O3PUFA against SCI-mediated chronic pain.

A polyamine-deficient diet opposes hyperalgesia, tolerance and the increased anxiety-like behaviour associated with heroin withdrawal in rats.

In humans, hyperalgesia, tolerance and anxiety disorders are common symptoms during heroin withdrawal syndrome. Significant evidence supports a role of NMDA receptors in these phenomena. Because polyamines may positively modulate the functioning of NMDA receptors and mainly originate from dietary intake, one hypothesis is that a polyamine deficient diet (PD diet) may reduce withdrawal symptoms. To address this question, we investigated the ability of a PD diet to prevent or to alleviate some symptoms of withdrawal syndrome as hyperalgesia, and increased anxiety-like behaviour in rats receiving 14 once daily subcutaneous heroin injections. Here, we show that a PD diet has both preventive and curative properties for reducing certain signs of withdrawal such as hyperalgesia, tolerance and increased anxiety-like behaviour observed in rats fed with a standard diet. Moreover, in heroin-withdrawn rats which were returned to basal pain sensitivity level, hyperalgesia following acute analgesia induced by a single heroin dose was observed in heroin-treated rats fed with standard diet, not in rats fed with a PD diet. Similarly, a stress-induced hyperalgesia induced by a non-nociceptive environmental stress session was observed in heroin-treated rats fed with standard diet. In contrast, a stress-induced analgesia was observed in heroin-treated rats fed with a PD diet, as it was observed in non heroin-treated rats. Since a PD diet for several weeks did not induce appreciable side-effects in rats, these preclinical results suggest that a PD diet could be an effective strategy for improving the relief of certain negative emotional states of heroin withdrawal syndrome and to allow reducing other medications generally used, such as opioid maintenance drugs.

[From the physiology of pain to an antihyperalgesic nutritional approach]. / De la physiologie de la douleur à une approche nutritionnelle anti-hyperalgésique.

Pain is brought into play by a system which detects and transmits messages signaling pain. Opioids negatively modulate nociception, but paradoxically also contribute to hyperalgesia. A nutritional approach with low polyamine contents helps to reduce this phenomenon without the harmful side effects of antihyperalgesic drugs.

Polyamine deficient diet to relieve pain hypersensitivity.

There is a compelling body of evidence that N-methyl-d-aspartate receptors (NMDA-R) play a critical role in the development and maintenance of pain hypersensitivity. However, long-term treatments with NMDA-R antagonists are limited by unacceptable side effects. Since polyamines modulate the functioning of NMDA-R and mainly originate from normal dietary intake and bacterial metabolism in the gut, we developed a nutritional therapy based on dietary polyamine deficiency. Here, we reported that a polyamine deficient diet (PD diet) for 7 days prevented the enhancement of tyrosine phosphorylation of the spinal NR2B subunit-containing NMDA-R associated with inflammation in rats. Based on these data, we studied the ability of PD diet to prevent long-lasting pain hypersensitivity associated with tissue injury on one hind paw by evaluating long-lasting changes in both mechanical nociceptive threshold and weight bearing. A PD diet strongly reduced long-lasting hyperalgesia induced by inflammation or incision, especially in fentanyl-treated rats. Moreover a PD diet also prevented the exaggerated hyperalgesia induced by a second inflammation performed 7 days after the first one. A PD diet also opposed paradoxical hyperalgesia induced by non-nociceptive environmental stress in rats with pain and opioid experiences. A PD diet reversed pain hypersensitivity associated with monoarthritis or neuropathy and restored the analgesic effect of morphine. Since PD diet was devoid of any noticeable side effects, this nutritional therapy could be part of an effective and safe strategy for pre-emptive analgesia and for reducing the transition from acute to chronic pain and its outcomes in various pain syndromes.

An evaluation of a polyamine-deficient diet for the treatment of inflammatory pain.

Polyamines are thought to be involved in the regulation of numerous metabolic and electrophysiological processes in the nervous system. In this study we evaluated the effect of a synthetic polyamine-deficient diet on pain in a carrageenan (Car)-induced inflammatory rat model. Inflammation was induced with a unilateral subcutaneous injection of Car in a plantar hindpaw in rats fed without (control group) or with (deficiency group) a polyamine-deficient diet. Ipsilateral and contralateral hyperalgesia was evaluated using the Randall-Sellito pressure test. Heart rate changes were also recorded under general anesthesia. Then, the effects of a bupivacaine sciatic nerve block and subcutaneous injection of naloxone or ketamine were evaluated for Car-induced hyperalgesia. Data were analyzed using analysis of variance followed by unpaired Student's t-test (significance P < 0.05). Before Car injection, no significant difference was observed in response to mechanical stimuli between the control and the deficiency groups (n = 114 in pooled data). Car injection induced significant ipsilateral and contralateral hyperalgesia in the control groups, whereas a significant analgesic effect appeared in the deficient groups on both the ipsilateral and contralateral hindpaws. This analgesic effect was confirmed by the electrocardiogram recording that showed a significant increase in heart rate in the control group after Car injection compared with the deficiency group that showed a decrease in heart rate under general anesthesia. Bupivacaine sciatic nerve block had no significant effect on hypoalgesia phenomena induced by polyamine deficiency. Naloxone administration had no effect in the control group but reversed the analgesic effect in the deficiency group. Ketamine administration induced a significant analgesic effect in the control group and partly reversed the analgesic effect in the deficiency group. In conclusion, a synthetic polyamine-deficient diet had a significant general analgesic effect on Car-induced mechanical hyperalgesia. The mechanism of analgesic action remains to be elucidated.

Consumption of soy diet before nerve injury preempts the development of neuropathic pain in rats.

BACKGROUND: A previous report using a partial sciatic nerve ligation (PSL) model for neuropathic pain in rats demonstrated that consumption of soy-containing diets preoperatively and postoperatively suppressed development of mechanical and heat allodynia, as well as hyperalgesia. The current study examined whether dietary soy suppresses these neuropathic sensory disorders when consumed either before or after PSL injury. METHODS: Male Wistar rats were grouped into seven different feeding regimens. These rats were fed SOY (RMH-1000, PMI Feeds, St. Louis, MO), a diet containing 85% soy protein since weaning, and were then switched to noSOY (Bio-Serv Co., Frenchtown, NJ), a diet devoid of soy at certain time points before PSL injury (14, 7, 1 days, or 15 and 0 h). Postoperatively, these rats were fed SOY or noSOY diets. Levels of mechanical and heat allodynia and hyperalgesia were determined preoperatively and 3, 8, and 14 days after PSL injury. RESULTS: Compared with groups fed preoperative noSOY, consumption of SOY before PSL injury significantly blunted postoperative levels of allodynia and hyperalgesia. Administering the SOY diet both before and after PSL injury provided no additional suppression of neuropathic pain. No pain suppression was noted in rats fed a noSOY diet preoperatively and SOY diet after PSL injury. Switching from SOY to noSOY feeding within 15 h of PSL injury was sufficient to allow for the full development of allodynia and hyperalgesia. CONCLUSIONS: Consumption of a soy-containing diet suppressed the development of neuropathic pain after PSL injury. The pain-suppressing properties of dietary soy were the result of a preemptive effect (i.e., when consumed preoperatively), but not a palliative effect (i.e., when consumed postoperatively). This effect of soy-containing diets appears to be short-lived, since switching to a noSOY diet 15 h before ligation abrogated the suppressive effect of soy.

Soy-containing diet suppresses chronic neuropathic sensory disorders in rats.

UNLABELLED: Partial sciatic nerve ligation (PSL) in rodents produces chronic neuropathic sensory disorders resembling neuropathic pain in humans. We previously reported that levels of allodynia and hyperalgesia after PSL injury were markedly attenuated by consumption of soy-containing diets. Here we aimed to show that dietary effect on pain behavior is not specific to a certain laboratory. For this purpose, experiments were conducted in a different laboratory (Baltimore rather than Jerusalem) and a different rat strain (Wistar rather than Sabra), with additional and different testing methods (radiant heat from a lamp rather than a CO(2) laser). Rats were fed two soy-free diets and a soy-containing one for 28 days. The sensitivity of rats to nonnoxious and noxious stimuli was determined before PSL injury, and levels of neuropathic sensory disorders were determined after it. We found that consuming the soy-containing diet prevented development of tactile and heat allodynia, but not mechanical hyperalgesia. This dietary effect was not correlated with calorie intake and weight gain or dietary concentration of fat and carbohydrates. We conclude that, regardless of experimental site, diet markedly affects chronic neuropathic sensory disorders in rats and should be standardized in animal models of pain. IMPLICATIONS: Levels of chronic sensory disorders in a rat model of allodynia and hyperalgesia after partial sciatic nerve ligation depend on the consumption of a soy-containing diet. Further studies are needed to determine the role of diet in humans with chronic pain.

Heat hyperalgesia following partial sciatic ligation in rats: interacting nature and nurture.

As in humans, levels of neuropathic pain produced by nerve injury are highly variable among animals. This variability was attributed to genetic and environmental factors. For example, we reported that chronic neuropathic sensory disorders developing following total (autotomy) or partial nerve injury (allodynia and hyperalgesia) depended on the diet rats consumed. Here we investigated the interaction between genetic and dietary factors in the development of heat hyperalgesia in rats following partial sciatic ligation (the PSL model). We show that heat sensitivity of intact rats and levels of heat hyperalgesia of PSL-injured rats were highly variable across eight different rat strains and seven different diets. Thus, genetic and environmental variables interact in determination of levels of chronic neuropathic sensory disorders in rats.