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2.3. Experimental Design

After the animals had acclimated for one week, sub-chronic exposure was performed. Fifty male SD rats (80 ± 20 g, 4-weeks-old) were divided randomly into five groups (with 10 rats in each groups), including control, Mn-treated, and three doses of PAS-Na (100, 200, and 300 mg/kg) treatment (Mn + 100, Mn + 200 and Mn + 300 PAS) groups. As in , the Mn-treated, Mn + 100, Mn + 200, and Mn + 300 PAS groups received intraperitoneal (i.p.) injections of MnCl2 (as 6.55 mg/kg Mn) once a day, five days per week, for 12 weeks, before subsequent treatment with physiological saline (for the Mn-treated group) and different doses of PAS-Na (100, 200, and 300 mg/kg, subcutaneous (s.c.) injections) in the back area of rats once a day, five days per week, for an additional six weeks. The control group received sterile physiological saline in the meantime. All of the i.p. and s.c. injections were given at volumes of 0.2 mL per 100 g rat weight. MnCl2 and PAS-Na were dissolved in sterile physiological saline each day before the injections.

3.1. PAS-Na Treatment Recovered Sub-Chronic Mn Exposure Induced Impairments of Spatial Learning and Memory Abilities

As shown in the spatial navigation test for assessing learning abilities, Mn-treated rats showed learning impairments that were characterized by an increased escape latency and swimming distance, when compared with those of the control group (repeated measures ANOVA F(4, 45) = 3.382, p = 0.017 for escaping latency; and F(4, 45) = 3.505, p = 0.014 for swimming distance (A,B). In contrast, PAS-Na treatments (100, 200, or 300 mg/kg) significantly improved the Mn-induced learning impairments, by decreasing the escape latency and the swimming distance (Tukey’s post-hoc test, p < 0.05 and p < 0.01 respectively, A,B). On the other hand, in the spatial probe trial for assessing memory, the number of platform crosses of the Mn-treated group was less than those of the control group (3.50 ± 1.96 vs. 6.20 ± 2.28 s, one-way ANOVA, F = 3.278, p = 0.021, C), suggesting memory deficits. As expected, PAS-Na treatment (300 mg/kg) also significantly attenuated the memory deficits induced by sub-chronic Mn exposure, restoring the number of platform crosses to control levels (multiple comparisons, p = 0.023, D). Furthermore, there was no difference observed in the swimming speed between different groups (one-way ANOVA, F = 0.538, p = 0.677, C), indicating that the aforementioned impairments by Mn did not result from possible muscular side effects of the Mn injections.

3.2. PAS-Na Treatment Failed to Restore Mn-Induced Decrease in GABA Levels in the Basal Ganglia

Next, we measured the GABA level in the basal ganglia to determine the possible involvement of GABA metabolism. Sub-chronic Mn exposure for 12 weeks and a six-week exposure cessation led to decreased GABA levels compared with the control [F(4, 45) = 4.337, p = 0.003, multiple comparisons, p = 0.02, ]. In contrast to the protective effects of PAS-Na in Mn-induced neurotoxicity, all doses of PAS-Na treatment for six weeks failed to affect the Mn-induced changes in the GABA levels (multiple comparisons, p > 0.05).

3.3. PAS-Na Treatment Restored Mn-Induced Changes of GABA Enzymes

To further investigate the effects of Mn on GABA metabolism, the enzymatic activities of GAD (a GABA-synthesizing enzyme) and GABA-T (a GABA-degrading enzyme) in the basal ganglia were analyzed. After Mn exposure for 12 weeks and a six-week exposure cessation, the GAD activity in the Mn-treated group was significantly increased compared with the control [F(4, 45) = 5.061, p = 0.003, and multiple comparisons, p = 0.0005, ]. In a similar pattern, the GABA-T activity of the Mn-treated group was significantly increased compared with the control [153% of control, F(4, 45) = 4.261, p = 0.004, and multiple comparisons, p = 0.0002, B]. In contrast, treatment with either 200 or 300 mg/kg PAS-Na for six weeks caused significant reductions in GAD activity, to 41.06% and 41.31% of the Mn-treated group, respectively(multiple comparisons, p = 0.005 or 0.009 respectively, A). PAS-Na treatment at the highest dose (300 mg/kg, but not 100 or 200 mg/kg) for six weeks also significantly reduced GABA-T activity by 24.0%, compared with the Mn-treated group (multiple comparisons, p = 0.005, B).

3.4. PAS-Na Prevented Mn-Induced Transcriptional Changes in GABAA and GAT-1 Expression

To investigate whether Mn affected GABA metabolism through the deregulation of its receptor or transporter, the mRNA and protein expressions of GABAA and GAT-1 in the basal ganglia were measured. Sub-chronic Mn exposure decreased GABAA mRNA expression in the basal ganglia [F(4, 20) = 12.182, p = 0.0008, multiple comparisons, p = 0.007, left panel of ], but significantly increased GAT-1 mRNA levels [F(4, 20) = 12.182, p = 0.003, multiple comparisons, p = 0.0002, right panel of ], when compared with the control. Treatment with 300 mg/kg PAS-Na (but not 100 and 200 mg/kg) for six weeks restored the above-mentioned changes by increasing GABAA mRNA expression (multiple comparisons, p = 0.033) and decreasing GAT-1 mRNA expression (multiple comparisons, p = 0.006). Although it seemed that sub-chronic Mn exposure increased GAT-1 protein expression and PAS-Na treatment could recover this, all of the changes failed to reach statistical significance (p > 0.05, ).

Abstract

A microporous hydrogel was developed using sodium alginate (alg) and 4-aminosalicylic acid (4-ASA). The synthesized hydrogel was characterized using various analytical techniques such as Fourier transform infrared spectroscopy (FTIR), Carbon-13 nuclear magnetic resonance (13C-NMR), X-ray powder diffraction (XRD), scanning electron microscopy (SEM), and differential scanning calorimetry (DSC). Additonal carboxyl and hydroxyl functional groups of 4-ASA provided significant lubrication and stress-triggered sol-gel transition to the conjugated hydrogel. In addition, cytotoxicity analysis was undertaken on the conjugated hydrogel using human dermal fibroblast-adult (HDFa) cells, displaying non-toxic characteristics. Drug release profiles displaying 49.6% in the first 8 h and 97.5% within 72 h, similar to the native polymer (42.8% in first 8 h and 90.1% within 72 h). Under applied external stimuli, the modified hydrogel displayed significant gelling properties and structure deformation/recovery behaviour, confirmed using rheological evaluation (viscosity and thixotropic area of 8095.3 mPas and 26.23%, respectively). The modified hydrogel, thus, offers great possibility for designing smart synovial fluids as a biomimetic aqueous lubricant for joint-related injuries and arthritis-induced conditions. In addtion, the combination of thixotropy, non-toxicity, and drug release capabilities enables potential viscosupplementation for clinical application.

1. Introduction

In the current study, we developed a novel microporous hydrogel based on sodium alginate and 4-ASA via carbodiimide coupling chemistry. This is the first report on the synthesis of an alginate-4-aminosalicylic acid based amide conjugate to the best of our knowledge. Furthermore, the modified hydrogel was found to be nontoxic and possesed drug release profiles simmilar to sodium alginate. Gelling ability and gel structure deformation/recovery behaviour was confirmed using rheological studies to evaluate the hydrogel as a stress-triggered hydrogel system. Development and evaluation of biopolymer-based soft hydrogel systems provide great possibilities for designing smart synovial fluids and may act as a biomimetic aqueous lubricant for arthritis treatment. Moreover, the combination of thixotropy, nontoxicity and drug release capabilities of the hydrogel enables significant potential for clinical application such as viscosupplementation in arthritis intervention.