Impact of altered environment and early postnatal methamphetamine exposure on serotonin levels in the rat hippocampus during adolescence.

BACKGROUND: Methamphetamine (MA) is a highly abused psychostimulant across all age groups including pregnant women. Because developing brain is vulnerable by the action of drugs, or other noxious stimuli, the aim of our study was to examine the effect of early postnatal administration of MA alone or in combination with enriched environment (EE) and/or stress of separate housing, on the levels of serotonin (5HT) in the hippocampus of male rat pups at three stages of adolescence (postnatal day (PND) 28, 35 and 45). MA (5 mg/kg/ml) was administered subcutaneously (sc) to pups (direct administration), or via mothers' milk between PND1 and PND12 (indirect administration). Controls were exposed saline (SA). Pups were exposed to EE and/or to separation from the weaning till the end of the experiment. RESULTS: On PND 28, in sc-treated series, EE significantly increased the muted 5HT in SA pups after separation and restored the pronounced inhibition of 5HT by MA. No beneficial effect of EE was present in pups exposed to combination of MA and separation. 5HT development declined over time; EE, MA and separation had different effects on 5HT relative to adolescence stage. CONCLUSIONS: Present study shows that MA along with environment or housing affect 5HT levels, depending on both the age and the method of application (direct or indirect). These findings extend the knowledge on the effects of MA alone and in combination with different housing conditions on the developing brain and highlight the increased sensitivity to MA during the first few months after birth.

Skin temperature contribution to the decrease in withdrawal latency following chronic constriction injury.

BACKGROUND: Chronic constriction injury (CCI) is widely used as an animal neuropathic pain model. Neuropathic pain is considered to exist when withdrawal latency to thermal stimulation is decreased after inducing a CCI to the sciatic nerve. However, it is known that CCI leads to changes in skin temperature and that skin temperature can affect withdrawal latency. Aim of this study was to compare withdrawal latencies of constricted and contralateral hind limbs, to thermal stimulation, at the same artificially-induced skin temperatures. METHODS: Neuropathic pain was induced by four ligatures on the left sciatic nerve in adult male Wistar rats. Withdrawal latencies were measured from the 11th to 14th day after ligation, in different ambient temperatures, using the plantar test (Hargreaves method). By changing ambient we produced different hind limb skin temperatures. RESULTS: Our results show that (1) CCI cause an increase in skin temperature; (2) the withdrawal latency was inversely related to ambient and skin temperature in the same manner for both the ligated and contralateral hind limbs; and (3) withdrawal latencies did not differ significantly for the ligated and contralateral hind limbs when the temperature of the hind limbs was artificially made the same (i.e., by changing the ambient temperature). CONCLUSIONS: Withdrawal latencies to thermal stimulation did not differ on ligated and contralateral hind limb after CCI to the sciatic nerve if the temperature of the hind limbs was artificially or mathematically made the same. This finding may have significant impact on the interpretation results of neuropathic pain research.

Separation of anaesthetic ketamine and its derivates in PAMAPTAC coated capillaries with tuneable counter-current electroosmotic flow.

Capillary electrophoretic separation of ketamine, norketamine, hydroxynorketamine, and dehydronorketamine was performed in the counter-current regime under the influence of oppositely-directed electroosmotic flow. For this purpose, the fused silica capillaries were covalently coated with the poly(acrylamide-co-3-acrylamidopropyl trimethylammonium chloride) copolymer (PAMAPTAC). The content of the cationic monomer APTAC in the polymerization mixture varied in the range 0-6 mol. % and the generated electroosmotic flow increased continuously in the 0-20 · 10-9 m2V-1s-1 interval. Importantly, it resulted in improved electrophoretic resolution of ketamine/norketamine, which increased from 0.8 for neutral PAM coating (i.e. 0% PAMAPTAC) to 3.0 for 6% PAMAPTAC. The determination of ketamine and its derivates in rat serum was performed in a 4% PAMAPTAC capillary with an inner diameter of 25 µm. The separation was performed in a 500 mM aqueous solution of acetic acid (pH 2.3). The clinical sample was deproteinized by the addition of acetonitrile to the serum and a large volume of the treated sample was injected directly into the capillary. The achieved limit of detection ranged from 2.2 ng/mL for dehydronorketamine to 4.1 ng/mL for hydroxynorketamine; the intra-day repeatability was 1.0-1.5% for the migration time and 2.8-3.3% for the peak area. The developed methodology was employed for time monitoring of ketamines in rat serum after intra venous administration of low doses of anaesthetic at a level of 2 µg per g of body weight.

Rapid electrophoretic monitoring of the anaesthetic ketamine and its metabolite norketamine in rat blood using a contactless conductivity detector to study the pharmacokinetics.

A method of capillary electrophoresis with contactless conductivity detection has been developed for non-enantioselective monitoring the anaesthetic ketamine and its main metabolite norketamine. The separation is performed in a 15 µm capillary with an overall length of 31.5 cm and length to detector of 18 cm; inner surface of the capillary is covered with a commercial coating solution to reduce the electroosmotic flow. In an optimised background electrolyte with composition 2 M acetic acid + 1% v/v coating solution under application of a high voltage of 30 kV, the migration time is 97.1 s for ketamine and 95.8 s for norketamine, with an electrophoretic resolution of 1.2. The attained detection limit was 83 ng/mL (0.3 µmol/L) for ketamine and 75 ng/mL (0.3 µmol/L) for norketamine; the number of theoretic plates for separation of an equimolar model mixture with a concentration of 2 µg/mL was 683 500 plates/m for ketamine and 695 400 plates/m for norketamine. Laboratory preparation of rat blood plasma is based on mixing 10 µL of plasma with 30 µL of acidified acetonitrile, followed by centrifugation. A pharmacokinetic study demonstrated an exponential decrease in the plasma concentration of ketamine after intravenous application and much slower kinetics for intraperitoneal application.

Poly(d,l-lactide)/polyethylene glycol micro/nanofiber mats as paclitaxel-eluting carriers: preparation and characterization of fibers, in vitro drug release, antiangiogenic activity and tumor recurrence prevention.

Poly(d,l-lactide)/polyethylene glycol (PLA/PEG) micro/nanofibers loaded with paclitaxel (PTX, 10 wt%) were prepared by needless electrospinning technology, which allows large scale production for real medicinal practice. The fiber structure and properties were investigated by several methods including scanning electron microscopy, nitrogen adsorption/desorption isotherm measurements, differential scanning calorimetry, and X-ray diffraction measurements to examine their morphology (fiber diameter distribution, specific surface area, and total pore volume), composition, drug-loading efficiency, and physical state. An HPLC-UV method was optimized and validated to quantify in vitro PTX release into PBS. The results showed that the addition of PEG into PLA fibers promoted the release of higher amounts of hydrophobic PTX over prolonged time periods compared to fibers without PEG. An in vitro cell assay demonstrated the biocompatibility of PLA/PEG fibrous materials and showed significant cytotoxicity of PTX-loaded PLA/PEG fibers against a human fibrosarcoma HT1080 cell line. The chick chorioallantoic membrane assay proved that PTX-loaded fibers exhibited antiangiogenic activity, with a pronounced effect in the case of the PEG-containing fibers. In vivo evaluation of PTX-loaded PLA/PEG fibers in a human fibrosarcoma recurrence model showed statistically significant inhibition in tumor incidence and growth after primary tumor resection compared to other treatment groups.

Electrophoretic large volume sample stacking for sensitive determination of the anti-microbial agent pentamidine in rat plasma for pharmacological studies.

A sensitive capillary electrophoretic method with on-line sample preconcentration by large volume sample stacking has been developed for determination of the anti-microbial agent pentamidine. The separation is performed in a fused silica capillary coated with covalently bound hydroxypropyl cellulose, with an internal diameter of 50 µm and length of 31.5 cm; the background electrolyte was 100 mM acetic acid/Tris at pH 4.7. The stacking is tested using a model sample of 1 µM pentamidine dissolved in 25% infusion solution and 75% acidified acetonitrile. Stacking permits the injection of a sample zone with a length of 95% of the total capillary length to achieve an enhancing factor of 77 compared to low injection into 1.8% of the total capillary length, with simultaneous high separation efficiency of approximately 1 350 000 plates/m. Stacking is based on simultaneous application of a separation field and a hydrodynamic pressure to force the acetonitrile zone out of the capillary. This approach allows the determination of pentamidine in rat blood plasma using only 12.5 µL of plasma treated by the addition of acetonitrile in a ratio of 1:3 v/v. The attained LOD is 0.03 µM and the intra-day repeatability is 0.1% for the migration time and 1.0% for the peak area at the injection 28.3% of capillary length. The performed pharmacokinetic study with ten-second scanning of the blood reveals rapid dynamics of pentamidine in the arterial bloodstream, while the changes are much slower in the venous system.

Mouse glutamate carboxypeptidase II (GCPII) has a similar enzyme activity and inhibition profile but a different tissue distribution to human GCPII.

Glutamate carboxypeptidase II (GCPII), also known as prostate-specific membrane antigen (PSMA) or folate hydrolase, is a metallopeptidase expressed predominantly in the human brain and prostate. GCPII expression is considerably increased in prostate carcinoma, and the enzyme also participates in glutamate excitotoxicity in the brain. Therefore, GCPII represents an important diagnostic marker of prostate cancer progression and a putative target for the treatment of both prostate cancer and neuronal disorders associated with glutamate excitotoxicity. For the development of novel therapeutics, mouse models are widely used. However, although mouse GCPII activity has been characterized, a detailed comparison of the enzymatic activity and tissue distribution of the mouse and human GCPII orthologs remains lacking. In this study, we prepared extracellular mouse GCPII and compared it with human GCPII. We found that mouse GCPII possesses lower catalytic efficiency but similar substrate specificity compared with the human protein. Using a panel of GCPII inhibitors, we discovered that inhibition constants are generally similar for mouse and human GCPII. Furthermore, we observed highest expression of GCPII protein in the mouse kidney, brain, and salivary glands. Importantly, we did not detect GCPII in the mouse prostate. Our data suggest that the differences in enzymatic activity and inhibition profile are rather small; therefore, mouse GCPII can approximate human GCPII in drug development and testing. On the other hand, significant differences in GCPII tissue expression must be taken into account when developing novel GCPII-based anticancer and therapeutic methods, including targeted anticancer drug delivery systems, and when using mice as a model organism.

Enhanced activity of hormone sensitive lipase (HSL) in mesenteric but not epididymal fat correlates with higher production of epinephrine in mesenteric adipocytes in rat model of cachectic rheumatoid arthritis.

Cachectic rheumatoid arthritis, the less frequent form of the disease, is associated with loss of fat mass and often more severe course of the disease. Its experimental model represents rat adjuvant arthritis (AA) characterized by edema, lack of appetite, sharp body weight and fat loss. As individual fat depots display functional differences, here we studied lipolytic activity and sensitivity to lipolytic stimuli of nodeless epididymal fat (eWAT) and perinodal mesenteric fat (mWAT) depots at the peak of AA. We also examined changes in catecholamine and cytokine levels involved in lipolysis in plasma and/or isolated adipocytes from both WATs to identify the contribution of local, adipocyte-based processes and/or systemic events to adiposity loss in cachectic rheumatoid arthritis. AA was induced to male Lewis rats by complete Freund's adjuvant. Groups of ad libitum-fed and pair-fed controls were used to distinguish the effects of food restriction from inflammation-induced cachexia. Adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and its phosphorylated form (pHSL) were analyzed by western blot. CRP and catecholamine levels in plasma or adipocyte lysates were determined using ELISA kits. Cytokine-induced neutrophil chemoattractant-1 (CINC-1/CXCL1), monocyte chemoattractant protein-1 (MCP-1/CCL2), IL-1ß, IL-6, IL-10 and leptin in adipocyte lysate were analyzed by quantitative protein microarray. Plasma glycerol and FFA were measured spectrophotometrically. AA rats developed severe cachexia, with lower adiposity in mWAT compared to normal and pair-fed controls, whereas in eWAT the adiposity was similarly reduced in AA and pair-fed groups. ATGL levels in both WATs were not affected by AA or pair feeding. AA upregulated levels of HSL, pHSL and pHSL/HSL ratio in mWAT, whereas none of these parameters has changed in eWAT of AA rats or in either WATs of pair-fed rats. In AA rats plasma glycerol was elevated, whereas FFA concentration was reduced. Plasma norepinephrine and epinephrine were increased in AA compared with both groups of controls. In eWAT adipocytes, AA but not pair feeding, upregulated norepinephrine levels. In mWAT adipocytes, AA rats showed higher epinephrine levels than pair-fed controls. Leptin levels in both WATs were depleted in AA animals in accordance with body weight loss. None of the measured cytokines in eWAT and mWAT was enhanced. Our results demonstrate augmented lipolytic activity in mWAT and not eWAT during cachectic arthritis. The adipocyte-derived cytokines do not seem to contribute to activated lipolysis. We first demonstrated enhanced presence of norepinephrine in perinodal adipocytes that may contribute to the regulation of local lipolytic activity by auto/paracrine fashion and thus provide independent fuel supply to activated lymph nodes.

Role of oxidative stress in animal model of visceral pain.

Reactive oxygen species play an important role both in physiological and pathophysiological reactions. The aim of this study was to determine the role of free radicals and antioxidants in the development of visceral pain. Visceral pain was produced by colorectal distension (CRD) in adult rats. CRD was caused by insertion of a lubricated latex balloon into the descending colon and rectum followed by inflation to 80mm Hg for 10min. During CRD, visceral pain was rated on 0-3.5 point scale. Oxidative stress was determined indirectly by measurement of free radical scavenging enzymes (glutathione peroxidase (GPx) and superoxide dismutase (SOD)) in the blood, liver and brain. Following CRD we observed (1) all rats expressed signs of visceral pain (overall rating was 1.83), (2) SOD and GPx levels were increased in the liver and blood, and decreased in the brain samples and (3) administration of the antioxidant Trolox, a water-soluble derivate of vitamin E, prior to CRD, prevented SOD and GPx changes in the liver, blood and brain, but did not affect pain scores. It was concluded, that CRD as a model of visceral pain, increases oxidative stress in animals, which could be prevented by prior administration of antioxidants; however, antioxidants did not attenuate signs of visceral pain caused by CRD.

Cortical stimulation and tooth pulp evoked potentials in rats: a model of direct anti-nociception.

While the effect of cortex stimulation on pain control is widely accepted, its physiological basis remains poorly understood. We chose an animal model of pain to study the influence of sensorimotor cortex stimulation on tooth pulp stimulation evoked potentials (TPEPs). Fifteen awake rats implanted with tooth pulp, cerebral cortex, and digastric muscle electrodes were divided into three groups, receiving 60 Hz, 40 Hz and no cortical stimulation, respectively. TPEPs were recorded before, one, three and five hours after continuous stimulation. We observed an inverse relationship between TPEP amplitude and latency with increasing tooth pulp stimulation. The amplitudes of the early components of TPEPs increased and their latency decreased with increasing tooth pulp stimulation intensity. Cortical stimulation decreased the amplitude of TPEPs; however, neither the latencies of TPEPs nor the jaw-opening reflex were changed after cortical stimulation. The decrease in amplitude of TPEPs after cortical stimulation may reflect its anti-nociceptive effect.

Effect of prenatal and postnatal methamphetamine exposure on nociception in adult female rats.

The aim of the present study was to determine effects of methamphetamine (MA) exposure and cross-fostering on thermal nociceptive thresholds in different estrous phases in adult female rats. Rat mothers were exposed daily to injection of MA (5 mg/kg) or saline for 9 weeks: prior to impregnation, throughout gestation and lactation periods. Dams without any injections were used as an absolute control. On postnatal day 1, pups were cross-fostered so that each mother raised four pups of her own and eight pups from the mothers with the other two treatments. Offspring females were tested in adulthood (85-90 days) for thermal nociception as latency [s] of withdrawal reaction of forelimbs, hind limbs, and tail. Our results showed that prenatal MA exposure did not affect the nociception in adulthood, while postnatal MA exposure (i.e., MA administration to lactating mothers) had pro-nociceptive effects. The effect of postnatal MA exposure was apparent in both, fore- and hind limbs, while the latency to tail withdrawal reaction was the same among the groups. In addition, the pro-nociceptive effect of postnatal MA exposure did not depend on estrous cycle. This study indicates that postnatal but not prenatal exposure to MA affects nociception in adult female rats. However, it is still not clear whether the pro-nociceptive effect of postnatal MA exposure is linked to direct action of MA on neuronal organization, or to indirect action of MA mediated by impaired maternal care.

Role of GABA(B) receptor agonist baclofen in acute pain modulation during the early postnatal period.

BACKGROUND: Baclofen, a specific GABA(B) receptor agonist, is used to treat spasticity and its off-label use includes the treatment of pain. The aim of this study was to show the role of baclofen in acute pain modulation during the early postnatal period. METHODS: Baclofen was tested in 2 doses (1 and 5 mg/kg) in 3 age groups (postnatal days 7, 16 and 21) in order to assess its effect on acute nociception and motor co ordination in rat pups. Pain was evaluated by measurement of paw/tail withdrawal latencies, and motor coordination by age-relevant tests. RESULTS: Although baclofen impaired motor function in all age groups, no changes in paw withdrawal latencies were observed. On the other hand, baclofen significantly increased tail withdrawal latencies of all groups. CONCLUSION: It is concluded that the GABA(B) system is functionally matured in the early postnatal period and plays a similar, but not the same, role as in adulthood.

Motor cortex stimulation in rats with chronic constriction injury.

Motor cortex stimulation (MCS) has gained a significant role in treatment of neuropathic pain. In order to evaluate effect of MCS in experimental animals we applied MCS to rats with neuropathic pain, which was evoked by chronic constriction injury (CCI) to the left sciatic nerve. Pain thresholds of both hind limbs were measured before, immediately after MCS, 1 h after MCS and 1 day after MCS. Effect of the stimulation was studied with respect to laterality (contralateral and ipsilateral MCS) and duration (short-term 10-min and long-term 1-h stimulation). It was found out that in control rats MCS did not affect thermal nociceptive thresholds. However, in CCI animals following results were obtained: difference score (difference in paw withdrawal latency between ligated and non-ligated hind limb) significantly decreased after both short- and long-term contralateral MCS; the difference score after the long-term ipsilateral MCS (related to the ligated hind limb) was not significantly different from that of intact animals; the effects of the contralateral short-term and the ipsilateral long-term stimulation faded within 1 h after the end of MCS, while the effect of the contralateral long-term MCS remained 1 h after the end of the MCS and faded within 24 h. It is concluded that MCS in experimental animals exerts similar effects as in human suffering from neuropathic pain and that the effect might be evoked from both cerebral cortices.

Different transfer of nociceptive sensitivity from rats with postnatal hippocampal lesions to control rats.

Hippocampal lesions in newborn rats alter the development of mechanisms involved in the processing of nociception. The hippocampal lesion was induced by the bilateral infusion, into the lateral cerebral ventricles, of 0.25 microL of saline containing either 0.25 micromol quinolinic acid (QUIN) and/or 0.25 micromol N-acetyl-L-aspartyl-L-glutamate (NAAG) on postnatal day 12. The same amount of sterile saline was injected into the sham-operated animals (group SHAM). It was expected that the QUIN- and NAAG-lesioned rats would exhibit some differences in thermal pain perception; however, we wanted to know if the control rats would exhibit, at least in part, similar changes in pain perception as their chemically lesioned siblings with which they were housed. Young adult NAAG-injured rats exhibited increased withdrawal latencies in the tail-flick and plantar tests, whereas young adult QUIN-injured animals exhibited only marginally decreased latencies. Nociceptive responses in the SHAM rats paralleled the littermates that had been neonatally treated with QUIN or NAAG, i.e. the responses in the SHAM(QUIN) group decreased, whereas the responses in the SHAM(NAAG) group increased. No significant changes in nociception were observed in intact animals, regardless of which group they were housed with. Our results show that social factors, which were originally demonstrated only for the pain behavior, may also influence basal nociceptive sensitivity in rats. We concluded that the "sham operation" may have had a long-term, nonspecific impact on nociceptive behavior by inducing behavioral mimicry of other animals.

The effect of motor cortex stimulation in deafferentated rats.

OBJECTIVES: The aim of the study was to describe the effect of motor cortex stimulation (MCS) on pain thresholds in deafferentated rats. SETTINGS AND DESIGN: The effect of MCS was studied in 18 deafferentated and 14 intact laboratory rats, using a standardised plantar test and tail-flick latencies. Two inoxious stimulation electrodes were implanted subdurally over the cerebral cortex and a C5-Th1 dorsal root rhizotomy was performed on the left side. Pain thresholds were measured before and after cortical stimulation. The data were analysed with ANOVA for repeated measures. RESULTS: MCS in intact animals evoked no changes in pain thresholds except for the contralateral forelimb, in which the pain threshold increased after MCS. Following deafferentation, pain thresholds increased in both plantar test and tail-flick in comparison to baseline values. When MCS was applied to the deafferentated animals, the pain thresholds returned to baseline levels. The effect of MCS disappeared within 24 hours. MAIN FINDINGS: 1. MCS in intact animals evoked hypoesthesia in the corresponding contralateral forelimb; 2. deafferentation itself increased pain thresholds in the unaffected limbs; 3. under MCS, pain thresholds in deafferentated rats were not different from pre-dafferentation values; 4. the effect of MCS disappeared in 24 hours and oscillated. CONCLUSIONS: Our results show a similar effect of the stimulation in man and experimental animals despite the differences in the organisation of the cerebral cortex. The use of laboratory animals is promising for further studies in the field of involved antalgic mechanisms of MCS.

Self-mutilation in young rats after dorsal rhizotomy.

OBJECTIVES: The aim of the study was to describe the development of self-mutilation after extensive dorsal rhizotomy of the brachial plexus performed during early ontogeny in rats. SETTINGS AND DESIGN: The rhizotomy was performed in three groups of rats according to the central nervous system maturation: infant, young, and adult. After the surgery the occurrence of self-mutilation behavior was compared. Rats from the infant group and non-mutilating deafferentated rats from the adult group underwent extracellular recordings from intralaminar thalamic neurons. Interspikes intervals of the records were compared by means of chaodynamic methods. RESULTS: In the infant group self-mutilation did not develop at all. Among the young group self-mutilation developed in 40% of rats and consisted of superficial wounds in all cases. In adult self-mutilation appeared in 80% rats and consisted of both superficial wounds (75%) and amputation (25%). In the newborn group and the deafferentated adult group without any signs of self-mutilation means of the parameters were not significantly different and were significantly lower than those of intact adult rats. MAIN FINDINGS: 1. Self-mutilation does not develop after the rhizotomy in the infant rats. 2. Neurons behave in chaotic way in adult as well as in young animals. 3. Chaodynamic parameters do not differ between infant and adult rats without any signs of self-mutilation. CONCLUSIONS: The results suggest that development of self-mutilation behavior in rats strongly depends on the ontogenetical period of nervous system injury, and that mature nervous system is required for the development of described pathological behavior.

Paradoxical firing of thalamic neurons under neuropathic pain state in rats.

OBJECTIVES: A novel evaluative approach was used to determine single unit activities of non-bursting intralaminar thalamic nuclei under neuropathic pain state following dorsal rhizotomy. SETTINGS AND DESIGN: Extensive dorsal rhizotomy at cervicothoracic level in rats was used as a model of central pain. After rhizotomy, rats were divided into two groups: rats without any signs of self-mutilation, and those presenting self-mutilation. Spontaneous single unit activities of neurons of intralaminar thalamic nuclei were recorded and interspike intervals (ISIs) of non-bursting cells were counted for both groups and compared with that of non-rhizotomized control rats. Chaodynamic methods were applied for the evaluation of the ISIs. RESULTS: In control rats Lyapunov exponents, Shannon entropy and mutual information average values were significantly higher than those of rhizotomized rats without any signs of self-mutilation. Paradoxically, in animals presenting self-mutilation following rhizotomy the evaluated parameters were similar to those of controls. Further, Lyapunov exponents were positive values in all animals indicating chaotic pattern of the neuronal firing. MAIN FINDINGS: 1. Neurons behave in chaotic way in all animals, 2. The most regular firing was found in non-mutilating rhizotomized animals, 3. Patterns of the firing in selfmutilating rats were similar to those in controls. CONCLUSIONS: It is concluded that pain feeling is not executed neither by changes of chaotic dynamics of non-bursting intralaminar thalamic neurons. On the other hand, the paradoxical firing of the neurons under pathological brain matrix might participate in modification pain feeling.