Transcriptome analysis of dorsal root ganglia's diabetic neuropathy reveals mechanisms involved in pain and regeneration
Introduction
Diabetic neuropathy (DN) is the most common complication of diabetes and one of the major causes of non-traumatic limb amputations in the US (Centers for Disease Control and Prevention. 2011 national diabetes fact Sheet). DN is a frequent condition, affecting 60% of diabetic patients [1], and, in the US, the estimated costs related to this complication ranges from 4,6 to 13,7 billion dollars [2].
Clinically, DN presents typical neuropathic symptoms, mainly related to impairment in sensory nerves, such as allodynia, spontaneous pain or no pain at all, accompanied by loss of sensitivity [3]. Once installed, DN presents maladaptive morphofunctional characteristics such as length-dependent axonal loss, affecting limb distal parts first [1,4].
Although DN is most often related with severity and duration of diabetes, its onset and how it is triggered remains unknown. Metabolic abnormalities interrelated and resulting from hyperglycemic environment, insulin and/or C peptide signaling impairment could have a role in diabetic neuropathy. However, the great number of cases refractory to treatments targeting those affected pathways and the poor correlation between development of DN and glycemic control suggests for other factors not associated with glucose regulation [5,6].
This way, we used a rat model for type 1 diabetes, treated with multiple low-doses of streptozotocin, and accompanied disease progression for 4 weeks in order to trace the earlier peripheral neuropathic symptoms, in an attempt to detect the first molecular alterations accompanying these sensorial changes. In order to check that, we used an electronic analgesimeter Von Frey to detect the modifications in mechanical sensitivity. Subsequently, L4 and L5 DRGs were collected to perform differential expression analysis by Next-Generation Sequencing (RNA-Seq). Although very similar, the transcriptome profile of the control group (C) and the diabetic neuropathy group (DN) showed some genes differentially expressed in this early phase of the disease. Thus, these alterations may consist of new mechanisms modified by hyperglycemia that could produce new insights for the understanding of the development of DN.
Section snippets
Animals
Male Wistar rats aged 7–10 weeks were obtained from the Multidisciplinary Center for Biological Investigation on Laboratory Animal Science (CEMIB) and the present research study protocol was approved by the University of Campinas' Animal Use Ethics Committee (CEUA) (Permit number 2658-1). Effort and care were taken in order to minimize animal suffering and to minimize the number of animals used. Rats were housed in a controlled environment with a photoperiod of 12 h and had full access to
Metabolic parameters
Blood glucose level at baseline was below 150 mg/dL, consistent to basal values obtained before STZ injection, at day 0 (Fig. 1A). The control group presented measurable physiological glucose levels throughout the duration of the experiment. On the other hand, the DN group, which received STZ i.p. injections for five days, started to present plasmatic glucose levels above the hyperglycemic threshold, 250 mg/dL, by day 7. This increase continued throughout the experiment, reaching the glucometer
Discussion
This is the first study to use RNA-Seq as a tool for prospecting transcriptome changes in DRGs of a rat model of type I diabetes experiencing the first symptoms of peripheral mechanical sensitization. Here we show that changes in mechanical sensitization could be detected two weeks after the first STZ injection, as a probable complication of systemic hyperglycemia. Our behavioral results matched previous studies [14,15], in which alterations in sensorial sensitivity were detected 1 to 3 weeks
Conclusions
Our data shows that during the first weeks after development of diabetic neuropathy, the DRG transcription profile of the diabetic group differs from the control group, which means that alterations due to hyperglycemic state are taking place in DRG cells. Functional categorization of genes showed alteration in the expression of genes mainly related to inflammation, hyperalgesia or analgesia, cell proliferation and apoptosis/cell survival, most of them never correlated to diabetic neuropathy
Abbreviations
- DN
diabetic neuropathy
- DRG
dorsal root ganglia
- STZ
streptozotocin
- RNA-Seq
Next-Generation Sequencing
- i.p.
intraperitoneal
Acknowledgements
We wish to thank the Central Laboratory of High Performance Technologies (LaCTAD) for the preparation of cDNA libraries and running the RNA-seq.
Competing interests
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Authors contributions
Conceived and designed the experiments: MCPA, ASV, CRS, CAP. Performed the experiments: MCPA, EVD, JMT, GGS. Analyzed the data: MCPA, ASV, CRS, CHT, CAP. Wrote the paper: MCPA, ASV, CAP.
Funding
This work was supported by São Paulo Foundation (Fundação de Amparo à Pesquisa Do Estado de São Paulo - FAPESP - http://www.fapesp.br/en/): Grant number - 2011/23764-0; Grant number - 2014/25153-7.
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