Elsevier

Life Sciences

Volume 205, 15 July 2018, Pages 54-62
Life Sciences

Transcriptome analysis of dorsal root ganglia's diabetic neuropathy reveals mechanisms involved in pain and regeneration

https://doi.org/10.1016/j.lfs.2018.05.016Get rights and content

Abstract

Peripheral diabetic neuropathy (DN) manifests in nearly 60% of diabetic patients, being pain its most debilitating symptom. Although electrophysiological and morphological aspects are well described, little is known about its development and progression, undermining effective therapies. Hyperglycemia and insulin signaling impairment are considered the triggering events of oxidative stress observed in the dying nerves, however there are still many gaps in the knowledge of intracellular plastic changes it generates.

Aims

In this study we aimed to evaluate the early transcriptome changes in DN when the first symptoms of the disease start to appear.

Main methods

Next-Generation Sequencing of messenger RNA (RNA-Seq) of L4 and L5 dorsal root ganglia (DRG) four weeks post-diabetes induction in a rat model for type 1 diabetes.

Key findings

RNA sequencing found 66 transcripts differentially expressed between diabetic and control groups, related mainly to the following biological processes: inflammation, hyperalgesia/analgesia, cell growth and cell survival. Given their roles, the differentially expressed genes suggest an attempt to switch to a survival/regenerative program.

Significance

Our results show that changes in the transcriptome profile start to appear early in the course of DN and might be related to secure cell homeostasis. Hence, the present data may indicate how DRG cells are responding to hyperglycemia in its early stages and which mechanisms first fail to respond, further leading to cell damage and cell death. Early screening of cell alterations in DN might lead to more concrete targets for pharmaceutical interventions, which could more efficiently delay cell damage.

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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