Effects of low-level laser therapy (LLLT) in experimental diabetic neuropathy

Author(s)

Willians Fernando Vieira

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2020-02-28

Examining board members

Carlos Amílcar Parada; Gisele Picolo; Rodrigo Antunes de Vasconcelos; Carmen Veríssima Ferreira; Marucia Chacur

• Advisor: Helder Jose Ceragioli; Carlos Amílcar Parada

Abstract

Peripheral diabetic neuropathy (PDN) occurs in 60 % of the diabetic patients, and it is considered the most common cause for neuropathic pain in the western world. Pain is the most common symptom of PDN, and the occurrence of gait alterations is also very frequent, which includes plantar pressure impairments. There is a strong correlation between the development of PDN and the increase in the concentration of proinflammatory cytokines, such as IL-1? and TNF-? in the dorsal root ganglia (DRG). These cytokines are linked to the activation of mitogen activated protein kinases (MAPKs) cascade, including p38, ERK1/2, and c-Jun-N-terminal proteins. There is no efficient treatment for NDP at a global level, except the rigorous hyperglycemia control. Among other non-pharmacological therapies, the Low-Level Laser Therapy (LLLT) could be an interesting option for the PDN treatment. However, there is little information about the influence of LLLT on the modulation of signaling pathways related to the PDN development and maintenance. Based on that, the aim of this study was to investigate the anti-hyperalgesic effect mechanisms of LLLT applied in streptozotocin (STZ)-induced diabetic neuropathic Lewis rats. All the experiments were approved by the UNICAMP Ethics Committee (CEUA), protocols n. 3902-1 and 5337-1/2019. Male isogenic Lewis rats, 200-250 g, 4-8-week-old, received 5 low-doses of STZ, 1 dose per day, during 5 consecutive days (25 mg/kg/day), or STZ vehicle, 0.1 M sodium buffer citrate. Type-1 diabetes was determined by the occurrence of glycemia ? 250 mg/dL at 48 h after the last STZ injection. Diabetic and control rats were submitted to Randall-Selitto, electronic von Frey, and CatWalk tests, at 0, 7, 14, 21, 24, and 28 days after the beginning of STZ or vehicle injections. Diabetic hyperalgesic rats were submitted to LLLT (GaAs, 904 nm; 2.03 J; 70 mW; 29 s), applied over the rats shaved skin at the dorsal region of L4-L5 DRG, bilaterally, between the 21st and the 28th days of the experimental protocol. At the 28th day, rats were euthanized and the L4-L5 DRG were collected and processed for ELISA, RT-qPCR, Western blot, immunofluorescence, and Raman spectroscopy analyses. In parallel, DRG from naïve rats were used for primary cell culture of afferent neurons, which were kept during 24 h under low- (5.5 mM of glucose) or high-glucose (55 mM of glucose) cell media, and used for calcium imaging, mitochondrial membrane potential (MMP), neuronal membrane potential through FluoVoltTM, patch clamp whole-cell and MTT cell viability assay. LLLT treatment was able to reduce the intensity of mechanical hyperalgesia in diabetic neuropathic rats, and to restore altered spatial gait parameters, strongly correlated to hyperalgesia [Print area (cm²); Max. contact area (cm²); Stride length (cm)]. LLLT treatment reduced the levels of DRG IL-1? and TNF-?, and the p38 MAPK mRNA expression. PDN was accompanied by the p38 MAPK phosphorylation, which was partially reduced by LLLT. Altered peaks from DRG Raman spectra of diabetic neuropathic rats (2704 cm-1; 2850 cm-1; 2885 cm-1; 3160 cm-1) showed normal intensities after LLLT treatment. DRG neurons kept in hyperglycemic medium and exposed to LLLT, showed reduced calcium influx and increased MMP, related to mitochondrial complexes I and III. There was no significant influence over the neuronal membrane potential. LLLT did not affect the cell viability of DRG neurons. In general, there were beneficial effects of LLLT in the treatment of PDN, once the therapy was able to act, positively, over behavioral, cellular, molecular, and spectral alterations (AU)

• FAPESP's process: 15/12673-5 - Effects of low-level laser therapy (LLLT) in experimental diabetic neuropathy
• Grantee: Willians Fernando Vieira
• Support Opportunities: Scholarships in Brazil - Doctorate