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.

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.

Characterization of the part of N-terminal PIP2 binding site of the TRPM1 channel.

Transient receptor potential melastatin-1 (TRPM1) is a calcium channel that is essential for the depolarization of photo-responsive retinal bipolar cells, but most of the physiological functions and cellular roles of this channel are still poorly understood. Most transient receptor potential (TRP) channels are typically regulated by intracellular proteins and other signaling molecules. Phosphatidylinositol-4,5 bisphosphate (PIP2), a minor phospholipid component of cell membranes, has previously been shown to directly bind TRP channels and to play a unique role in modulating receptor function. To characterize the binding of PIP2 as a potential regulator of TRPM1, we utilized biophysical methods and molecular modeling to study the interactions of PIP2 with an N-terminal fragment of TRPM1 (residues A451-N566). The basic N-terminal residue K464 of TRPM1 suggests that it is part of putative pleckstrin homology (PH) domain and is involved in the interactions with PIP2. This is the first report detailing the binding of PIP2 at the N-terminus of the TRPM1 receptor.

PIP2 and PIP3 interact with N-terminus region of TRPM4 channel.

The transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective ion channel broadly expressed in a variety of tissues. Receptor has been identified as a crucial modulator of numerous calcium dependent mechanisms in the cell such as immune response, cardiac conduction, neurotransmission and insulin secretion. It is known that phosphoinositide lipids (PIPs) play a unique role in the regulation of TRP channel function. However the molecular mechanism of this process is still unknown. We characterized the binding site of PIP2 and its structural analogue PIP3 in the E733-W772 proximal region of the TRPM4 N-terminus via biophysical and molecular modeling methods. The specific positions R755 and R767 in this domain were identified as being important for interactions with PIP2/PIP3 ligands. Their mutations caused a partial loss of PIP2/PIP3 binding specificity. The interaction of PIP3 with TRPM4 channels has never been described before. These findings provide new insight into the ligand binding domains of the TRPM4 channel.

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.

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.

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.