Scorpion (Hottentotta tamulus) venom pre-exposure delays functional recovery in mice following peripheral nerve injury

Abstract

Scorpion sting leads to profound challenges of central nervous system (CNS) impairments such as neuro-inflammation, unconsciousness, aberrant ion channels physiology, epilepsy and may become fatal due to heart failure. However persistence of Hottentotta tamulus venom in peripheral nerves and subsequent influence on regenerative process of injured peripheral nerve remains unknown. Current study reports the persistence of H. tamulus venom components 30-days following its intraperitoneal administration in sciatic nerves (SN) of mice pre-exposed through either a single-toxin exposure (STE) or multiple-toxin exposure (MTE). Of note, venom pre-exposure delays and compromises the sensori-motor functional recovery in STE and MTE mice following standard sciatic nerve crush injury. Histological investigations of regenerating SN and gastrocnemius muscles (GCM) 14-days post crush injury exhibited reduced myelination and limited numbers of motor axons in SNs and GCM of MTE mice, respectively. Consistently, a marked reduction in expression of regeneration-promoting markers including transcription factors (such as Atf-3 and c-Jun), regeneration associated genes (such as Sprr1a and Gap-43) and ion channel proteins encoding genes (such as Scn9a and Kcc2) was observed in lumber dorsal root ganglia (DRG) and regenerating SN 14-days post crush injury. Collectively, this study reports the persistence and regeneration-inhibiting effects of H. tamulus venom in peripheral nerve of pre-exposed mice leading to compromised functional recovery.

Fig 1. Scorpion envenomation delayed the functional recovery onset in mice following sciatic nerve crush injury.

A. A delayed onset of sensory functional recovery in mice treated with single toxin exposure (STE) group (8dpi) and multi toxin exposure (MTE) mice (13dpi) comparing to control (CL) mice (7dpi), led to compromised full regain of sensory functions till the end of study duration (29dpi). B & C. Motor functional recovery in STE and MTE mice groups was hindered as depicted in lower toe spreading and more negative SFI scores and it was delayed comparing to CL mice. n = 5-6per group, data shown as mean±SEM. *p < 0.05 CL vs STE; #p < 0.05, ###p < 0.001 CL vs MTE. Two-way ANOVA with Bonferroni post-hoc analysis.

Fig 2. Scorpion venom exposure limits regenerative capacities of injured sciatic nerve.

A. Histological observations of regenerating SN revealed a limited remyelination in MTE mice. Scalebar 5µm. B. Quantitative estimation of myelination revealed a significant reduction in MTE group comparing to either CL or STE mice. C. A higher number of regenerating motor axons in silver nitrate stained GCM sections were observed in CL and STE groups, however a limited number of motor axons were seen in MTE mice GCM sections. Scalebar 100µm. Data represented as mean ±SEM; n = 3mice/group; ***p < 0.001, CL vs MTE; ###p < 0.001 STE vs MTE. One-way ANOVA followed by Bonferroni post-hoc analysis.

Fig 3. Scorpion envenomation reduces the regenerative capacities of injured peripheral neurons and sciatic nerves through modulating expression of regeneration marker genes and ion channel encoding genes 14dpi.

Atf-3 and c-Jun are down-regulated in lumber DRGs. Gap-43 and Sprr1a are downregulated in MTE mice while in STE only Gap-43 is significantly downregulated, comparing to CL. Na ion channel encoding gene Scn9a is downregulated in STE, while K ⁺ /Cl⁻ cotransporter encoding gene Kcc2 is downregulated in MTE mice. In regenerating SNs, a significant downregulation of Atf-3 was observed in STE and MTE, while expression levels of c-Jun fluctuated non-significantly. Similarly, the expression of Gap-43 was downregulated in STE and MTE mice in SNs, while the levels of Sprr1a were fluctuated non-significantly in venom-injected mice groups. Data represented as mean±SEM, n = 3mice per group. *p < 0.05; **p < 0.01; ***p < 0.001 CL vs venom-injected groups. Oneway ANOVA followed with Bonferroni posthoc analysis.