Pretreatment tumor prostaglandin E2 concentration and cyclooxygenase-2 expression are not associated with the response of canine naturally occurring invasive urinary bladder cancer to cyclooxygenase inhibitor therapy

The purpose of this study was to determine the extent to which pretreatment prostaglandin E2 (PGE2) concentration and cyclooxygenase-2 (cox-2) expression could be used to predict the antitumor activity of cox inhibitor treatment in naturally occurring canine transitional cell carcinoma of the urinary bladder (TCC). Snap frozen tissues (to measure PGE2) and formalin-fixed TCC samples (for cox-2 immunohistochemistry) were obtained by cystoscopy or surgery. Complete tumor staging was performed before and after one month of treatment with the cox inhibitor, piroxicam (0.3 mg/kg q24 h po). The pretreatment PGE2 concentration ranged from 57 to 1624 ng/g of TCC tissue; n=18 dogs). Cox-2 immunoreactivity was observed in all TCC samples. There was no association between PGE2 concentration, cox-2 expression, and change in tumor volume with piroxicam treatment. In conclusion, cox-2 expression or PGE2 concentration alone, or the combination of the two was not useful in predicting response to piroxicam treatment in canine TCC.     

Cyclooxygenase-2 expression in astrocytes and microglia in human oligodendroglioma and astrocytoma

Cyclooxygenases (cox) are potent mediators of inflamation and two cox-izoenzymes, cox-1, cox-2, are described to date. Cox-2 is cytokine-inducible in inflammatory cells and enhanced cox-2 expression has been attributed a key role in the development of edema and immunomodulation in pathologically altered brain tissues. In normal cerebral cortex cox-2 is present only in neurons, but not in the glial or vascular endothelial cells. The function of microglia in glioma biology is unclear. Microglia have both neurotrophic and neurotoxic functions and have been shown to release a variety of cytokines. Our preliminary results showed that the expression pattern of cox-2 is predominantly neuronal although glial expression was observed with the correlation of high malignancy. In this study we aimed to assess the phenotypes (astrocyte, microglia) of the cox-2-expressing glial cells in various types of human gliomas and to compare their expression patterns. For this purpose we employed dual immunohistochemistry for cox-2 and GFAP (astrocyte) or LCA-MAC (microglia-macrophage) in archival formalin-fixed, paraffin embedded human tissue diagnosed as oligodendroglioma and/or astrocytoma. The results showed that cox-2 immunoreactivity is up-regulated in the neurons according to the tumor grade. Most of the cox-2 immunoreactive glia were GFAP-positive in anaplastic oligodendrogliomas and at lesser extend in glioblastomas. Cox-2 and LCA co-localization was detected in more glial cells in glioblastomas. It may be speculated that the induction of cox-2 in microglia may contribute to the deleterious effects of prostanoids in cerebral edema formation during the progression of oligodendrogliomas. The detection of cox-2 in astrocytes surrounding the necrotic areas might be important to develop new strategies, such as the usage of cox-2 inhibitors combine with chemotherapy and radiotherapy in the treatment of glioma patients.     

Expression of cyclooxygenase-1 and 2 in naturally-occurring canine cancer

The purpose of this work was to determine cox-1 and cox-2 expression by immunohistochemistry in forms of naturally occurring canine cancer in order to identify animal systems for pre-clinical evaluation of cox inhibitors and cox-2 inhibitors in cancer. Canine lymphoma (LSA), prostatic carcinoma (PCA), osteosarcoma (OSA), oral melanoma (MEL), oral squamous cell carcinoma (SCC), oral fibrosarcoma (FSA), mammary carcinoma (MCA), and normal tissues were included. Cox-2 was expressed in epithelial tumors (17 of 26 SCC, 8 of 13 MCA, 5 of 9 PCA cases) and MEL (9 of 15 cases), but was generally absent in normal tissues. Cox-2 expression was minimal or absent in mesenchymal tumors and LSA. Cox-1 was expressed in normal epithelial tissues and in some osteoclast and osteoblast in bone, but was absent in normal lymph node. In conclusion, forms of canine cancer were identified for in vivo studies of the effects of cox inhibitors and selective cox-2 inhibitors on cancer.     

In vivo inhibition of cyclooxygenase-2 by a selective phosphorothioated oligonucleotide

Inhibition of cyclooxygenase-2 (cox-2) is considered to be anti-inflammatory, whereas inhibition of the constitutive isozyme cox-1 causes renal and gastrointestinal toxicity. Therefore, to achieve an optimal anti-inflammatory effect, an inhibitor should be cox-2 selective without inhibiting cox-1. For this purpose, 10 different cox-2-selective phosphorothioated oligonucleotides (S-oligos) were tested to inhibit the cox-2 enzyme selectively in vivo. An aqueous solution of these S-oligos (3 mg/kg body weight) was injected intraperitoneally (i.p.) into male Sprague-Dawley rats with colitis induced by trinitrobenzene sulfonic acid (TNBS). The colonic levels of cox-2 protein, mRNA, myeloperoxidase (MPO), and prostaglandin E2 (PGE2) were increased significantly on day 1 and remained significantly elevated until day 7 post-TNBS administration, whereas cox-1 remained unaltered. Two S-oligos were found to be effective in reducing the level of cox-2 protein selectively without any effect on the cox-1. The effective S-oligo, but not the mismatched control oligo, reduced the tissue levels of PGE2 and MPO activity significantly. The effective S-oligo reduced the level of cox-2 but not the cox-1 mRNA significantly, whereas a mismatched or a sense control oligo did not affect the levels of these isoforms. M-fold analysis demonstrated extensive secondary structure formation in the cox-2 mRNA. These findings demonstrate that only a few selected sites in the cox-2 target mRNA are accessible in vivo, probably because of the presence of secondary structures. Suppression of cox-2 protein, PGE2, and MPO activity by the S-oligo might prove to be an anti-inflammatory property.     

Cyclooxygenase-2 is expressed by cumulus cells during oocyte maturation in cattle.

Prostaglandins could be involved in various aspects of final differentiation of ovarian follicles. Prostaglandins are generated by the cyclooxygenase (cox) pathway. Until now, the expression pattern of isoforms cox-1 and cox-2 of cyclooxygenase in bovine cumulus-oocyte complexes (COCs) was unknown. Using immunodetection procedure, we demonstrated in the present study that cox-2 was expressed by cumulus cells during in vivo and in vitro maturation. Time course induction of cox-2 expression was investigated during in vitro maturation using Western blot analysis. Specific signal of cox-2 was markedly evidenced from 6 hr of culture and increased to reach a maximal level at 24 hr of culture. In vitro, cox-2 expression in COCs was associated with increased concentrations of PGE(2) and PGF(2alpha) in the maturation medium. In addition, the effects of culture conditions on cox-2 expression was considered using RT-PCR and Western-blot analysis. We demonstrated that the addition of 10 ng/ml of EGF to TCM199 clearly increased the expression level of cox-2 mRNA and protein. Higher levels of in vitro cox-2 expression was associated with greater rates of cumulus expansion and oocytes at metaphase II at 24 hr of culture. In conclusion, our present results suggest that cox-2 expression in cumulus cells may be involved in differentiation of COCs that occurs during oocyte maturation.     

Effect of cyclooxygenase inhibitors on the vasopressin induced ACTH and corticosterone response during crowding stress.

The aim of the present study was to compare the effect of social stress on the corticotropin releasing hormone (CRH) and arginine vasopressin (AVP)-induced pituitary-adrenocortical activity. Also the significance of prostaglandins (PG) generated by constitutive and inducible cyclooxygenase (COX-1 and COX-2) in the stimulation of hypothalamic-pituitary-adrenal (HPA) axis by AVP under basal and crowding stress conditions was investigated. The control rats were housed 7 in a standard cage and stressed rats were crowded 24 in a cage of the same size during 7 days. The activity of HPA axis was determined by measuring plasma ACTH and serum corticosterone levels 1 h after i.p. AVP administration. Indomethacin (2.0 mg/kg i.p.), a non-selective COX inhibitor, piroxicam (0.2, 2.0, and 5.0 mg/kg), a more potent COX-1 than COX-2 inhibitor, and compound NS-398 (0.2 and 2.0 mg/kg) a selective COX-2 inhibitor, were administered i.p. 15 min prior to AVP (5.0 microg/kg i.p.) to control or crowded rats. The obtained results indicate that social stress for 7 days considerably inhibits the stimulatory action of AVP on ACTH secretion, while it intensifies the CRH-induced ACTH secretion. Indomethacin, piroxicam and NS-398 significantly diminished the AVP-elicited ACTH and corticosterone secretion in non-stressed rats. None of these COX antagonist induced any significant inhibition of the AVP-induced ACTH and corticosterone secretion in stressed rats. Therefore, PG generated by COX-1 or COX-2 do not participate to a significant extent in the HPA stimulation by AVP during crowding stress. These results suggest that social crowding stress desensitizes the PG stimulatory mechanism which considerably mediates the AVP-induced HPA stimulation under basal conditions. The results contrast with a lack of any involvement of PG in the CRH-induced stimulation of HPA response under basal or crowding stress conditions.     

Mucosal damage induced by preferential COX-1 and COX-2 inhibitors: role of prostaglandins and inflammatory response

Nonsteroidal anti-inflammatory drugs (NSAID) are well known to induce gastric mucosal damage including bleeding, ulceration and perforation in humans and animals too. These effects are related with the inhibition of the enzyme cyclooxygenase, which is the main established mechanism of action for these drugs. Fasted rats were given piroxicam, preferential COX-1 inhibitor (10-20 mg/kg) or meloxicam, preferential COX-2 inhibitor (7.5-15 mg/kg) orally. Six or nine hours (h) later, respectively, the stomach was excised, the severity of the damage assessed and myeloperoxidase (MPO) activity measured, as well as prostaglandin PGE(2) content. Furthermore, in order to assess the effects of these oxicams over previously damaged gastric mucosa, 1 ml of 0.6 N HCl was administered p.o. followed, 1 h after, of the correspondent dose of each NSAID, and the same parameters were determined. Oral administration of both drugs dose-dependently caused acute gastric haemorrhage erosions. Myeloperoxidase activity was significantly increased by piroxicam administration. In addition, PGE(2) content was significantly reduced. The association between the administration of the acid and NSAID caused a worsening of the damage and, while myeloperoxidase activity did not modify by both piroxicam and meloxicam, PGE(2) levels were reduced. These results suggest that the PG derived from both COX-1 and COX-2 pathway plays a beneficial role in the gastroprotection, and thus caution should be exercise in the clinical use of preferential COX-2 inhibitors.     

Role of prostaglandins and nitric oxide in the lipopolysaccharide-induced ACTH and corticosterone response.

This study was designed to determine the role of endogenous prostaglandins (PG) and nitric oxide (NO) in the lipopolysaccharide (LPS)-induced ACTH and corticosterone secretion in conscious rats. LPS (0.5 and 1 mg/kg) given i.p. stimulated the hypothalamic-pituitary-adrenocortical (HPA) activity measured 2 h later. A non-selective cyclooxygenase inhibitor indomethacin (10 mg/kg i.p.), piroxicam (2 mg/kg i.p.), a more potent antagonist of constitutive cyclooxygenase (COX-1) and compound NS-398 (2 mg/kg i.p.), a selective inhibitor of inducible cyclooxygenase (COX-2) given 30 min before LPS (1 mg/kg i.p.) significantly diminished both the LPS-induced ACTH and corticosterone secretion. COX-2 blocker was the most potent inhibitor of ACTH secretion (72.3%). Nomega-nitro-L-arginine methyl ester (L-NAME 2 and 10 mg/kg i.p.), a non-selective nitric oxide synthase (NOS) blocker given 15 min before LPS did not substantially alter plasma ACTH and corticosterone levels 2 h later. Aminoguanidine (AG 100 mg/kg i.p.), a selective inducible nitric oxide synthase (iNOS) inhibitor, considerably enhanced ACTH and corticosterone secretion induced by a lower dose (0.5 mg/kg) of LPS and did not significantly alter this secretion after a larger dose (1 mg/kg) of LPS. L-NAME did not markedly affect the indomethacin-induced inhibition of ACTH and corticosterone response. By contrast, aminoguanidine abolished the indomethacin-induced reduction of ACTH and corticosterone secretion after LPS. These results indicate an opposite action of PG generated by cyclooxygenase and NO synthesized by iNOS in the LPS-induced HPA-response.     

Upregulation of macrophage migration inhibitory factor (MIF) and CD74, receptor for MIF, in rat bladder during persistent cyclophosphamide-induced inflammation

The objective of this study was to determine if macrophage migration inhibitory factor (MIF) is upregulated in the bladder during persistent cystitis. MIF is a pro-inflammatory cytokine found pre-formed in the urothelium. Previous findings showed that acute bladder inflammation increased MIF release into the bladder lumen while upregulating MIF and CD74 (MIF receptor) in the bladder. Because the effects of persistent cystitis on MIF and CD74 are not known, MIF and CD74 changes in the bladder were examined after short-term (1-day) or persistent (8-day) cyclophosphamide (CYP)-induced bladder inflammation. Anesthetized male Sprague-Dawley rats received either a single CYP treatment (150 mg/kg, ip; saline, control) and examined 1 day after treatment (short-term), or repeated CYP doses (20-75 mg/ kg, ip; saline, control; every third day for 8 days) and examined after 8 days of treatment (persistent). MIF protein levels in urine and bladder were determined. In addition, Mif, CD74, and cox-2 expression in the bladder was determined. Histology verified cystitis and MIF and CD74 immunoreactivity in the bladder. Repeated CYP doses were decreased to avoid toxicity. Short-term or repeated low CYP doses (40 mg/kg; 8 days) increased urinary MIF and decreased bladder MIF amounts while upregulating bladder Mif and CD74 mRNA expression. Persistent CYP-induced bladder inflammation (even at 40 mg/kg; 8-day treatment) also upregulated other inflammatory cytokines (CCL5, IL-11, iNOS) in the bladder. Short-term and persistent (low dose) CYP cystitis are associated with markedly increased MIF release into the urine and upregulation of Mif and CD74 in bladder. This supports the hypothesis that MIF and CD74 play a significant role in both acute and persistent stages of bladder inflammation.     

Envelope gene-mediated neurovirulence in feline immunodeficiency virus infection: induction of matrix metalloproteinases and neuronal injury

The release of neurotoxins by activated brain macrophages or microglia is one mechanism proposed to contribute to the development of neurological disease following infection by lentiviruses, including feline immunodeficiency virus (FIV). Since molecular diversity in the lentiviral envelope gene influences the expression of host molecules implicated in neuronal injury, the role of the envelope sequence in FIV neuropathogenesis was investigated by using the neurovirulent FIV strain V1CSF, the nonneurovirulent strain Petaluma, and a chimera (FIVCh) containing the V1CSF envelope gene in a Petaluma background. All three viruses replicated in primary feline macrophages with equal efficiency, but conditioned medium from V1CSF- or FIVCh-infected cells was significantly more neurotoxic than medium from Petaluma-infected cultures (P < 0.001) and could be attenuated in a dose-dependent manner by treatment with either the matrix metalloproteinase (MMP) inhibitor prinomastat (PMT) or function-blocking antibodies to MMP-2. Although FIV sequences were detectable by PCR in brain tissue from neonatal cats infected with each of the viral strains, immunohistochemistry revealed increased astrogliosis and macrophage activation in the brains of V1CSF- and FIVCh-infected cats relative to the other groups, together with elevated markers of neuronal stress that included morphological changes and increased c-fos immunoreactivity. Similarly, MMP-2, but not MMP-9, mRNA and protein expression was increased in brain tissues of V1CSF- and FIVCh-infected cats relative to Petaluma-infected animals (P < 0.01). Infection with V1CSF or FIVCh was also associated with greater CD4(+) cell depletion (P < 0.001) and neurodevelopmental delays (P < 0.005), than in Petaluma-infected animals; these deficits improved following PMT therapy. These findings indicated that diversity in the envelope gene sequence influenced the neurovirulence exhibited by FIV both in vitro and in vivo, possibly through a mechanism involving the differential induction of MMP-2.     

The CO2-SFE crude lipid extract and the free fatty acid extract from Perna canaliculus have anti-inflammatory effects on adjuvant-induced arthritis in rats

The anti-inflammatory (AI) activity of a supercritical fluid extract (CO(2)-SFE) of tartaric acid-stabilised Perna canaliculus mussel powder, and of the free fatty acid (FFA) class separated from the CO(2)-SFE extract by column chromatography, was investigated in the rat adjuvant arthritis model. Administration of the CO(2)-SFE extract (100 mg/kg BW/day s.c.) for 15 days post-adjuvant inoculation significantly reduced rear paw swelling by 34% and the deterioration in total body condition by 52% in arthritic rats, compared to vehicle controls. These observations were accompanied by a decreased serum ceruloplasmin oxidase activity, and reduced inflammatory response of the spleen. The mussel FFA extract given at one third of the dose (30 mg/kg BW/day s.c.) and for a shorter treatment period (5 days during the inflammatory phase) achieved an even greater AI activity, and was equipotent to piroxicam (2 mg/kg BW/day s.c.). Preliminary toxicology assessment using both arthritic and non-arthritic (healthy) rats revealed no significant differences between the mussel treatment groups and respective vehicle controls in either organ weights, tissue histology or selected biochemical parameters. These results indicate the CO(2)-SFE crude lipid extract and its FFA components from stabilised P. canaliculus mussel powder contain biologically significant AI activity in vivo, with no apparent adverse side effects.     

Participation of m- and n-cholinergic mechanisms in the development of the kindling phenomenon of the amygdala in cats

The kindling phenomenon was produced after chronic electrostimulation of the cat amygdala. The duration and intensity of petit mal and grand mal were recorded. The M-cholinomimetic arecoline (0.3 mg/kg), the acetylcholinesterase inhibitor galanthamine (1-3 mg/kg) and the N-cholinergic blockers eterofen (5-10 mg/kg) and ganglerone (035-3.5 mg/kg) intraperitoneally decreased or abolished the kindling phenomenon. Combination of M-cholinomimetics with N-blockers facilitated the anticonvulsant effects. Nicotine (0.5 mg/kg) and the M-cholinergic blocker methylbenactyzine (0.5-1 mg/kg) as well as combination of methylbenactyzine with galanthamine, on the contrary, facilitated and aggravated seizures in cats. In the authors' opinion, the M- and N-cholinergic mechanisms are involved in formation of the kindling phenomenon. It is suggested that N-cholinergic blockers or their combinations with M-cholinomimetics may be used as anticonvulsants.     

IL-6 activity in feline infectious peritonitis

Involvement of IL-6 in the development of vasculitis and polyclonal gammopathy in feline infectious peritonitis (FIP) was investigated, by using the proliferative responses of two IL-6-dependent murine hybridoma cell clones, B3B1 and MH60.BSF-2 cells. A significant IL-6 activity was found in sera and ascitic fluids of cats with FIP, whereas no IL-6 activity was detected in sera from healthy cats. In these FIP cats, IL-6 activity in ascitic fluids was significantly higher than that in sera. Peritoneal exudate cells from FIP cats were also found to release a high level of IL-6 to the culture supernatant. The ascitic IL-6 activity was eluted into the fractions corresponding to the m.w. of 30,000 to 40,000 in gel filtration, and into the fractions at the salt concentration from 0.2 to 0.3 M NaCl in anion exchange chromatography. The level of ascitic IL-6 activity was inversely correlated to serum albumin/globulin ratio in these FIP cats. These findings indicate that IL-6 accumulated in the ascites might leaked into the systemic circulation, and be linked to systemic alterations such as enhanced synthesis of Ig and acute phase proteins.     

Purification and partial characterization of feline alpha1-proteinase inhibitor (falpha1-PI) and the development and validation of a radioimmunoassay for the measurement of falpha1-PI in serum

Alpha(1)-proteinase inhibitor (alpha(1)-PI) of the domestic cat (Felis catus) was purified from serum and a radioimmunoassay (RIA) for the measurement of feline alpha(1)-PI concentration in serum was developed and validated. Feline alpha(1)-PI (falpha(1)-PI) was isolated using ammonium sulfate precipitation, anion-exchange, size-exclusion, ceramic hydroxyapatite, and hydrophobic interaction chromatography. The molecular weight of falpha(1)-PI was estimated at 57,000 and the relative molecular mass (M(r)) was determined to be approximately 54.5 kDa. Isoelectric focusing revealed four bands with isoelectric points (pI) between 4.3 and 4.5. The N-terminal amino acid sequence of the first 19 residues was Glu-Gly-Leu-Gln-Gly-Ala-Ala-Val-Gln-Glu-Thr-Val-Ala-Ser-Gln-His-Asp-Gln-Glu. Antiserum against feline alpha(1)-PI was raised in rabbits. Tracer was produced by iodination ((125)I) of feline alpha(1)-PI using the chloramine T method. A radioimmunoassay was established and validated by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and inter-assay variability. A control range for serum feline alpha(1)-PI concentration was established from 50 healthy cats using the central 95th percentile. The sensitivity of the assay was 0.042 mg/ml. Observed to expected ratios for serial dilutions ranged from 105% to 141.18% for four different serum samples at dilutions of 1 in 35,000, 1 in 70,000, 1 in 140,000 and 1 in 280,000. Observed to expected ratios for spiking recovery ranged from 88.14% to 152.17% for four different serum samples and five different spiking concentrations. Coefficients of variation for four different serum samples were 4.57%, 6.45%, 8.52%, and 4.27% for intra-assay variability and 6.88%, 9.57%, 7.44%, and 9.94% for inter-assay variability. The reference range was established as 0.25-0.6 mg/ml. In summary, feline alpha(1)-PI was successfully purified from serum using a rapid and efficient method. The radioimmunoassay described here is sensitive, linear, accurate, precise, and reproducible and will facilitate further studies of the physiological or potential pathological role of alpha(1)-PI in cats.     

Diminution of antifungal activity of fluconazole associated with ibuprofen and piroxicam in experimental histoplasmosis of hamsters (Mesocricetus auratus)

The aim of this study was to determine if tha association of non-steroid antiinflammatory drugs (piroxicam and ibuprofen) with fluconazole, affects the antifungal activity of the azole compound, in an experimental model histoplasmosis in hamsters (Mesocricetus auratus). Sixty hamsters were intracardially inoculated with 4x10(6) yeasts of Histoplasma capsulatum var. capsulatum. Treatments began one week after the challenge and continued for three weeks. The hamsters were divided in six groups of ten animals each and received the following treatment: 1- fluconazole 8 mg/kg/day; 2- ibuprofen 20 mg/kg/day; 3- piroxicam 20 mg/kg/day; 4- fluconazole+ibuprofen; 5- fluconazole+piroxicam and 6- only received the solvent of these drugs. One week after ending the treatment, all the animals were sacrified and the evaluation of the treatments was based on the results of blood cultures, on the determination of colony forming units per gram of spleen, and the histopathologic studies of the same organ. The animals treated with fluconazole plus ibuprofen or piroxicam showed more colony colony forming units per gram (3.9x10(7) and 3.3x10(7)) when compared with the animals treated with fluconazole alone (0.9x10(7)). The histopathologic results of the hamsters that received fluconazole showed well-organized granulomas with few yeast-like elements inside the macrophages. In contrast, those which received fluconazole associated with antiinflammatory drugs presented lax granulomas containing numerous yeast-like elements. These findings let us to conclude that non-steroids antiinflammatory drugs diminish the antifungal efficacy of fluconazole in this animal model.     

The Effects of Piroxicam in the Attenuation of MPP+/MPTP Toxicity In Vitro and In Vivo

Several lines of evidence support the neuroprotective action of cyclooxygenase-2 (COX-2) inhibitors in various models of Parkinson’s disease (PD). In the current study, we investigated the neuroprotective properties of several COX inhibitors against 1-methyl-4-phenylpyridinium (MPP+) in neuroblastoma Neuro 2A (N-2A) cells in vitro and the protection against degeneration of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons after the administration of 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) in C57/BL6 male mice. The data obtained demonstrate a lack of protective effects observed by COX 1-2 inhibitors ibuprofen and acetylsalicylic acid against MPP+ toxicity in N-2A, where piroxicam was protective in a dose dependent manner (MPP+ control: 15 ± 2% MPP+ piroxicam: 5 mM 89 ± 4%). The data also indicate a drop in mitochondrial oxygen (O₂) consumption and ATP during MPP+ toxicity with no restoration of mitochondrial function concurrent to a heightened concentration of somatic ATP during piroxicam rescue. These findings indicate that the neuroprotective effects of COX inhibitors against MPP+ are not consistent, but that piroxicam may work through an unique mechanism to propel anaerobic energy metabolism. On the other hand, using mice, piroxicam (20 mg/kg) was effective against MPTP-induced dopaminergic degeneration in the (SNc) and loss of locomotive function in mice. Administering a 3 day pre-treatment of piroxicam (20 mg/kg) was effective in antagonizing the losses in SNc tyrosine hydroxylase protein expression, SNc DA concentration and associated anomaly in ambulatory locomotor activity. It was concluded from these findings that piroxicam is unique among COX inhibitors in providing very significant neuroprotection against MPP+ in vitro and in vivo.     

Use of Arc expression as a molecular marker of increased postsynaptic 5-HT function after SSRI/5-HT1A receptor antagonist co-administration

An increase in central postsynaptic 5-hydroxytryptamine (5-HT) function activates expression of activity-related cytoskeletal protein (Arc). Here, Arc expression was used to test whether, in rats, co-administration of a 5-HT re-uptake inhibitor (paroxetine) and a 5-HT1A receptor antagonist (WAY 100635) increases postsynaptic 5-HT function. After pre-treatment with WAY 100635 (0.3 mg/kg s.c.), paroxetine (5 mg/kg s.c.) caused a threefold increase in 5-HT in prefrontal cortex microdialysates. In situ hybridization studies found that neither paroxetine (5 mg/kg s.c.) nor WAY 1000635 (0.3 mg/kg s.c.) altered Arc mRNA abundance in any region examined. In contrast, paroxetine (5 mg/kg s.c.) increased Arc mRNA after pre-treatment with WAY 100635 (0.3 mg/kg s.c.). This increase was apparent in cortical regions (frontal, parietal and cingulate) and caudate nucleus but was absent in hippocampus (CA1). Increases in Arc mRNA were accompanied by an increase in c-fos mRNA. The increase in Arc expression induced by paroxetine/WAY 100635 was abolished by the 5-HT synthesis inhibitor, p-chlorophenylalanine (300 mg/kg i.p., daily for two days). In conclusion, paroxetine and WAY 100635 injected in combination (but not alone) caused a region-specific, 5-HT-mediated increase in Arc expression. These data provide molecular evidence that co-administration of a 5-HT re-uptake inhibitor and 5-HT1A receptor antagonist increases 5-HT function at the postsynaptic level.     

Advantages of using same species enzyme for replacement therapy in a feline model of mucopolysaccharidosis type VI

In a feline model of mucopolysaccharidosis type VI (MPS VI), recombinant feline N-acetylgalactosamine-4-sulfatase (rf4S) administered at a dose of 1 mg/kg of body weight, altered the clinical course of the disease in two affected cats treated from birth. After 170 days of therapy, both cats were physically indistinguishable from normal cats with the exception of mild corneal clouding. Feline N-acetylgalactosamine-4-sulfatase was effective in reducing urinary glycosaminoglycan levels and lysosomal storage in all cell types examined except for corneal keratocytes and cartilage chondrocytes. In addition, skeletal pathology was nearly normalized as assessed by radiographic evidence and bone morphometric analysis. Comparison of results with a previous study in which recombinant human 4S (rh4S) was used at an equivalent dose and one 5 times higher indicated that rf4S had a more pronounced effect on reducing pathology than the same dose of rh4S, and in some instances such as bone pathology and lysosomal storage in aorta smooth muscle cells, it was as good as, or better than, the higher dose of rh4S. We conclude that in the feline MPS VI model the use of native or same species enzyme for enzyme replacement therapy has significant benefits.     

Effect of Chronic Administration of Low Dose Rapamycin on Development and Immunity in Young Rats

Mammalian target of rapamycin (mTOR) regulates cell growth, cell differentiation and protein synthesis. Rapamycin, an inhibitor of mTOR, has been widely used as an immunosuppressant and anti-cancer drug. Recently, mTOR inhibitors have also been reported to be a potential anti-epileptic drug, which may be effective when used in young patients with genetic epilepsy. Thus, a suitable dose of rapamycin which can maintain the normal function of mTOR and has fewer side effects ideally should be identified. In the present study, we first detected changes in marker proteins of mTOR signaling pathway during development. Then we determined the dose of rapamycin by treating rats of 2 weeks of age with different doses of rapamycin for 3 days and detected its effect on mTOR pathway. Young rats were then treated with a suitable dose of rapamycin for 4 weeks and the effect of rapamycin on mTOR, development and immunity were investigated. We found that the expression of the marker proteins of mTOR pathway was changed during development in brain hippocampus and neocortex. After 3 days of treanent, 0.03 mg/kg rapamycin had no effect on phospho-S6, whereas 0.1, 0.3, 1.0 and 3.0 mg/kg rapamycin inhibited phospho-S6 in a dose-dependent manner. However, only 1.0 mg/kg and 3.0 mg/kg rapamycin inhibited phospho-S6 after 4 weeks treatment of rapamycin. Parallel to this result, rats treated with 0.1 and 0.3 mg/kg rapamycin had no obvious adverse effects, whereas rats treated with 1.0 and 3.0 mg/kg rapamycin showed significant decreases in body, spleen and thymus weight. Additionally, rats treated with 1.0 and 3.0 mg/kg rapamycin exhibited cognitive impairment and anxiety as evident by maze and open field experiments. Furthermore, the content of IL-1β, IL-2, IFN-γ, TNF-α in serum and cerebral cortex were significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. The expression of DCX was also significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. However, rats treated with 1.0 mg/ kg rapamycin exhibited fewer and milder side effects than those treated with 3.0 mg/kg. In summary, all these data suggest that there is not a rapamycin dose that can inhibit mTOR for epilepsy without causing any side effects, but 1 mg /kg may be the optimal dose for young rats for suppressing mTOR with relatively few side effects.