MPTP had been characterised as a neurotoxin in humans and rodents, with irreversible Parkinson’s-like features such as tremor, rigidity, and slowness of movement (bradykinesia), postural instability, and freezing. The above motor symptoms were observed in the present study upon administration of MPTP, in validation of earlier reports by Aarslandet al. (2009), Jellinger(1998), and Meredith & Rademacher (2011), in which rigidity, slowness of movement, postural instability, and freezing were reported in MPTP-administered humans, non-human primates, cats, rabbits, and some rodents strains. Furthermore, this study further shows that BALB/c mice are also sensitive to MPTP with respect to these Parkinson’s-like features. However, these parkinsonic traits were absent in the mice pre-administered with Nigella sativa (NS + MPTP), indicating the prophylactic potential of Nigella sativa in preventing these Parkinson’s-like symptoms.
Upon macroscopic examination, no detectable insult or injury was observed in the brains across all groups. However, idiopathic scoliosis (IS) was observed in one of the MPTP-administered mice. Described as a multifactorial disease involving many intrinsic factors such as genetics, imbalance of muscle structures, abnormal growth of vertebral bodies, asymmetrical growth of the neurocentral cartilage, length discrepancy between spine and spinal cord, abnormal platelet calmodulin, and abnormality in melatonin metabolism (Banala et al., 2018; Machida, 2018); the idiopathic scoliosis observed in one of the MPTP mice may however be an epiphenomenon rather than being consequential to the MPTP administered, as has been said of many hypothesised factors (Machida, 2018).
Human PD patients have been reported to experience low body weight, a phenotype hypothesised to be predisposed by many factors like dysphagia, chewing difficulty, impaired hand-mouth coordination, and hyposmia (Bachmann & Trenkwalder, 2006; Ma et al., 2018). Similar body weight trend has also been reported of non-human primates (Porras et al., 2012). Despite this trend however, no significant weight loss was recorded in C57BL/6 mice by Sundström et al. (1990) during 4 weeks after MPTP exposure. A further obvious contrast to the trend above was the observable body weight changes in the current study, in which the MPTP mice had the highest mean body weight increase compared with all other groups.
While this may be explained by the increased food and water consumption rates observed in the mice, it is a further validation of the report in which 32.9% of PD patients studied by Cersosimo et al.(2018) showed increase in their body weights. This study thus shows MPTP as increasing MBW in BALB/c mice. The increased appetite and the consequent body weight gain also raises the question about the tendency of MPTP or perhaps some of its active constituents to to predispose exposed animals to obesity, as found in the BALB/c mice here studied. It may also be inferred that MPTP does not adversely affect the weight and that the growth rate is also normal in BALB/c’s mice, an effect that is yet to be understood.
However, the weight gain in the NS mice was significantly lower and in fact the least when compared to other groups. This is similarly explained by the feed consumption rate which was lower in the NS group than observed in all other groups .Reduced weight gain as recorded in this study, is a phenomenon that has been previously characterised to Nigella sativa exposure, in line with the findings of Bano et al. (2009) and Le et al. (2004), such that its significantly repressive effect on weight gain is obvious. This weight limiting effect of Nigella sativa was thus seen on the NS + MPTP mice which had a significantly lesser MBW than the MPTP group but higher than the NS group. Nigella sativa was thus able to limit the weight gain in this group, suggesting its ability to repress/counter the weight increasing effects of MPTP.
Relative brain weight (RBW), also known as brain-to-body weight ratio, is a hypothetical estimate of the brain functions of an animal, as it is believed to coincide much better with the observed cognitive abilities than absolute brain size (Cairό, 2011). In this study, the highest RBW was recorded in the MPTP mice, possibly due to a progression of the brain weight along with the body weights of the MPTP animals as discussed above. The NS + MPTP mice however recorded significantly lower RBW than the MPTP mice, indicating the impact of NS pre-exposure in keeping the RBW at par with the level recorded in the NS mice.
Novel object recognition test (NORT) is a measure of recognition memory which harnesses the innate tendency of mice to explore novelty of their environment (SBFNL(b), 2019). The MPTP mice showed the highest exploration time with the novel object in the assay, when compared with other groups, thus accruing the highest recognition memory to the MPTP mice. This finding is inconsistent with the previous reports in which mild cognitive impairment and dementia are established as pathophysiological features of Parkinson's disease at the early and later stages respectively (Weil et al., 2018). Recognition memory deficit particularly has been further documented by some studies in patients with Parkinson's disease (Chiaravalloti et al., 2014). The improved recognition memory found in this study may be explained by the higher brain-body weight ratio found in the MPTP mice, since the later, as earlier explained is hypothesised as a measure of brain function.
MPTP is a known neurotoxin with neurodegenerative potentials in human and animals (Langston, 2017). However, the memory and brain weight findings in the BALB/c mice administered with MPTP raises questions about possible advantageous impacts of this neurotoxin on some brain functions, even if such impacts are species or strain dependent.
The novel exploration time of the MPTP mice was significantly higher than recorded in the NS + MPTP mice, with the latter value similar to that of the NS mice. This may suggest the impact of Nigella sativa in moderating the novel object exploration time in the pre-treated mice.
The NS group behaved best by spending the least time to turn and the least time to reach the end of the beam. The MP animals however spent the longest time to reach the end of the beam, similar to the significantly (p < 0.0001) longer time reported of the Aphakia mice (also known as ak mouse, which is a striatal denervation model of Parkinson's disease exhibiting the cell specificity of neurodegeneration observed in humans) to complete the balance beam test (Singh et al., 2007). Nigella sativa was however able to reduce this traversing interval for NSMP but not the turning time. This suggests a therapeutic effect of Nigella sativa on motor balance and coordination in Parkinsonic mice.
The neurotransmitter assay revealed the lowest striatal dopamine in the MPTP mice, in validation of previous studies which characterised Parkinsonism with deficiency of dopamine and dopaminergic neurons in the midbrain, especially in the striatum (Scherman et al., 1989; Triarhou, 2013). The striatal dopamine levels in the NS and control group however were similar and both were significantly higher than in the MPTP mice. This may explain the up-regulated dopamine level recorded in the NS + MPTP mice in comparison with the parkinson’s-like MPTP mice. This indicates the prophylactic role of Nigella sativa in Parkinsonism. The cerebellar dopamine level in the MPTP mice was however higher than all other experimental groups, albeit insignificantly statistically, while in the frontal cortex, there was almost no difference in the dopamine levels across the mice. This corroborates the localisation of Parkinsonic dopamine depletion to the striatum (Folarin et al., 2019; Meredith & Rademacher, 2011; Scherman et al., 1989; Sedaghat et al., 2014; Singh et al., 2007).
The striatal neuronal density in the MPTP mice was the least, in validation of reports by Ross et al., (2004) where the mean neuronal densities in all striatal quadrants were significantly lower in the PD group compared with the other groups. This thus confirms the depletion of striatal neurons in BALB/c strains of MPTP mice models of Parkinsonism, in slight contrast to its being referred to as MPTP-resistant (Meredith & Rademacher, 2011). However, when pre-treated with Nigella sativa oil, striatal neuronal density was markedly preserved than in mice administered with MPTP only and Nigella sativa oil only.. The fact, however, that the NS + MPTP mice expressed more striatal neurons than the MPTP mice, corroborates the prophylactic potential of Nigella sativa against Parkinsonic endophenotypes as earlier described with other data above. Strains of mice are known to differ in their response to MPTP with respect to the degree of striatal DA depletion, amount of loss of midbrain DA neurons, and behavioural deficits. However, while the hallmark of sensitivity and resistance in the substantia nigra -pars compacta (SNpc) was put at “>50% SNpc neuron loss” and “<25% SNpc loss” respectively (Meredith & Rademacher, 2011), the neuronal loss recorded in the BALB/c MPTP mice was only significant statistically, but not up to 20% of the control’s neuronal density. A “>50% loss” was however recorded in the dopamine assays in the MPTP mice.
In the cerebellum and frontal cortex however, no significant morphological or pathological difference was observed between the groups across the molecular, ganglionic and granular layers of their cerebellar cortices; and across the layers of the frontal cortex.