Exploring the protective role of metformin and dehydrozingerone in sodium fluoride-induced neurotoxicity: evidence from prenatal rat models.

The findings of the present study demonstrated NaF induced oxidative damage, altering the behavior of animals, histopathology and neurochemical changes significantly. Treatment with metformin and DHZ decreased the oxidative stress and restored the antioxidant enzymes significantly. The behavioral and histopathological changes that occurred due to NaF treatment were restored in the treatment groups. This study reported the protective role of metformin and DHZ against 100 mg/L of NaF induced behavioral changes, histopathological changes and oxidative stress in brains of pre-natal rats.

Previous studies on metformin and DHZ reported multiple activities like antioxidant, anti-inflammatory and neuroprotective effects (Begum F et al. 2023). Clinical studies showed that chronic use of metformin among diabetic patients led to good cognitive function, when compared to patients who were on other anti-diabetic drugs (Kuo PC et al. 2005; Piwkowska et al. 2010; Rao MC et al. 2010; Park DB 2015; De Oliveira et al. 2016b; Dehkordi et al. 2019). Some studies have also shown that Metformin decreases AChE activity in the brain leading to restoration of normal memory and learning functions (Esteghamati et al. 2013; Saliu JA et al. 2016). Metformin and DHZ have been reported for their neuroprotective activity but there are no reports published on metformin and DHZ against exposure of NaF in prenatal pup’s developing brain. According to a study by Wang et al. in 2004, excess fluoride exposure led to diminished intelligence quotient in children (Wang J 2004). A study showed that NaF intoxication in rats led to memory loss and disability in learning (Ge Y et al. 2005). A study on pre-natal rats reported loss of memory and learning disability with increase in latency period in NaF group compared to normal control group pups (Mesram et al. 2017b).

Long term exposure to NaF decreases the spatial and episodic memory in rats (Liu et al. 2014). This study assessed the episodic memory using novel object recognition test. NaF exposure in disease group significantly reduced the recognition index i.e. ability of the rats to recognize novel object in presence of a familiar object during the test phase, as compared to the normal control group (at p<0.001). Recognition index in groups treated with Metformin and DHZ was significantly increased (at p<0.0001) as compared to NaF treated group. Metformin and DHZ treated rats showed significant improvement in discrimination index wherein both treatments showed equally significant increase. These results indicate loss of recognition and episodic memory in rats exposed to NaF during their developmental stages and reversal of this impairment due to treatment with both Metformin and DHZ.

NaF decreases the motor exploratory activity which is associated with decreased levels of AChE in brain (Vani et al. 2000a; Kumar et al. 2020). Variables like decreased locomotor activity, less time spent in central square, increased latency to first line crossing, increased rearing behaviour etc. provides a measure of anxiety in the animals (Sestakova et al. 2013; Oyagbemi et al. 2020) The parameters of motor exploratory activity viz., latency, grooming, rearing, sniffing, crossing was analyzed in the open field test and the locomotor activity was analyzed using Actophotometer. The study results indicate a decrease in locomotor activity and increased anxiety in rats exposed to NaF in pre-natal model and subsequent reversal due to treatment with Metformin (p<0.05). DHZ (p<0.01) was able to partially improve the impairments in group IV.

Latency to first line crossing was significantly elevated in disease group compared to the normal control group (p<0.01). Treatment with metformin (p<0.006) was able to reduce it significantly but the difference between DHZ group and disease was not significant (p<0.05).

NaF treatment in rats significantly increased AchE levels in hippocampus, however, increase in enzyme levels in frontal cortex was not found to be significant compared to the normal control group (p<0.05). Metformin has been studied for its action on AchE activity in streptozotocin (STZ) induced diabetes models. Studies reported that treatment with Metformin significantly reduced AchE levels in the brain. A similar observation was made in this study where elevation of AchE levels due to NaF exposure, was significantly reversed by Metformin & DHZ treatment (p<0.001) in hippocampus of rats. However in frontal cortex, the decrease in AchE levels was not found to be significant (p<0.05) (Saliu JA et al. 2016; Bhutada 2011) These results indicate elevated AchE enzyme levels could be involved in memory and locomotor impairments seen in various behavioural tests conducted and subsequent improvement due to treatment of Metformin and DHZ.

Lipid peroxidation (MDA levels) was significantly increased in hippocampus and treatment with Metformin was able to reduce this significantly. However, DHZ was unable to show a similar level of reduction and the difference between disease group and DHZ group was found to be non-significant at p<0.05. In frontal cortex, MDA levels were increased in disease group compared to normal control group and DHZ (p<0.01) decreased MDA levels significantly as compared to Metformin (p<0.01). Results obtained for reduced GSH levels were not similar for hippocampus and frontal cortex. In hippocampus, chronic exposure to NaF, declined GSH levels compared to the normal control group (p<0.01) and treatment with both Metformin and DHZ (p<0.001) was not able to significantly restore its levels. In frontal cortex, however, more promising results were observed and treatment with metformin (p<0.001) significantly increased GSH levels compared to disease group. DHZ did not show any significant changes in GSH levels.

Kumar et al. 2017 and Shanmugam et al. 2018 reported that exposure to sodium fluoride led to a reduction in body weight of rats (Shanmugam et al. 2018; Kumar et al. 2020). A similar observation was made in this study where, in the pre-natal model, a significant decrease in body weights of rats in disease group was observed compared to the normal control group (p<0.05). It was also observed that treatment with Metformin and DHZ was able to attenuate this loss in body weight significantly (at p<0.0001).

Histopathological studies revealed that the regions of the hippocampus and frontal cortex (Fig. 16), dentate gyrus (Fig. 15), and CA3 (Fig. 13) displayed a small number of degenerated neurons that were darkly stained (basophilic), with a shrunken and fragmented nucleus in the disease group (NaF only). In contrast, the normal control and treatment groups displayed neurons that were in good health, with a pale and round nucleus, a well-defined nuclear boundary, and prominent nucleoli. The deteriorated neurons in the CA3, dentate gyrus regions of the hippocampus, and frontal cortex of the normal control and treatment groups are less than those in the disease group (NaF alone). In contrast, CA1 (Fig. 14) in all groups had healthy neurons and did not exhibit many histological alterations.

An additional study was conducted for the rats in prenatal model in which the pups were tested for normal reflexes in early post-natal days (day 7–11) to check whether administration of NaF during gestation period had any effect on the reflexes and if treatment with metformin and DHZ were able to show any improvement in these reflexes. In the present study, no significant effect of NaF in prenatal rats was observed on any reflex. Also, treatment with Metformin and DHZ did not significantly improve the performance of pups for any reflex compared to both normal control and disease group. Thus, it can be concluded that NaF administration during gestation period does not have any effect on formation of reflexes in rat pups (Rao MC et al. 2011; Park DB 2015).

NaF exposure did not significantly impact neonatal reflexes, but it did impair cognitive function. Also led to decreased locomotor activity and increased anxiety-like behavior in pups. Metformin and DHZ treatments significantly improved cognitive deficits and reduced anxiety, with Metformin being particularly effective in restoring normal locomotor activity levels. Both Metformin and DHZ showed potential in mitigating the biochemical alterations, pressing on their antioxidative properties. Neuronal degeneration in the hippocampus of NaF-exposed rats, was effectively prevented by Metformin and DHZ treatment.