Factors Associated with Diabetic Neuropathy in Rats : a Systematic Review with Metanalysis

Diabetic neuropathy refers to a group of symptoms associated with the nerve’s damaged ability to perform its functions correctly. Symptoms may vary from paresis to pains that mainly affect sensitive or motor long nerves of feet and hands. Hence, experiments and analysis carried out with rodents, especially rats, allow several clinical interfaces to be known. Therefore, the aim of this study was to analyze the factors associated with diabetic neuropathy in rats. This is a systematic review with metanalysis. Data collection followed some prior election criteria. The sources of information for the researched data are the databases MEDLINE/PubMed and Scopus. The research was conducted using the keywords found in descriptors/MeSH: rats, diabetic neuropathies, and risk factors with the Boolean operator “AND”. The BioEstat 5.3 was used for statistical analysis, and the Odds Ratio was calculated with a 95% confidence interval (CI). Eighty-three references were found, of which 17 followed the criteria and were then included in the present review. Some factors like nerve morphology, blood hypertension, oxidative stress, diabetes period, hypoglycemic effect, vascular complications, and insulin parameters represent the main risk factors for the development of diabetic neuropathy, as well as diabetes induction in rats by using drugs. Such factors are very similar to those from humans, thus requiring a deeper analysis of the theme in a considerable human sample. Factors Associated with Diabetic Neuropathy in Rats: a Systematic Review with Metanalysis


Introduction
Diabetic neuropathy refers to a group of symptoms associated with the nerve's damaged ability to perform its functions correctly [1].Symptoms may vary from paresis to pains that mainly affect long nerves of feet and hands.This disease has been widely related to diabetes mellitus, especially when it is associated with high non-controlled glycemic levels [2].
The analysis of diseases in animals enables the association of new clinical and therapeutic guidelines in certain clinical conditions, since experimental studies are implemented more easily and answers to a certain conduct can be provided quicker for analysis [3].Hence, experiments and analysis carried out with rodents, especially rats, allow several clinical interfaces to be known [4].
Much has been described in literature about the morphological and morphometric alterations in spinal nerves, both in diabetic patients and in the experimental models of the disease.However, a comparison between these alterations and the eventual injuries seen in cranial and spinal nerves is being made, as well as a comparison with malnourished animals due to the regional basic diet (RBD) -a type of special diet that simulates human malnutrition in rats.Furthermore, it has been widely described in literature that diabetic patients show hearing disorders; however, it has not been found studies of an association with malnutrition or an association between diabetes and malnutrition.Despite the histological alterations of patients' internal ear structures and those in experimental models, a histological assessment of the vestibulocochlear nerve is being described in malnourished animals through the RBD for the first time.Firstly, the animals were chronically submitted to Streptozotocin (STZ)-induced experimental diabetes.Then, 42-day-old animals received a single dose injection of 60 mg/kg (STZ, Sigma ® ) intraperitoneally.Additionally, in the Control Group of the same age, rats received an injection of 60mg/kg of saline solution (citrate buffer) intraperitoneally.After 78 days of STZ inoculation, adult animals aged around 120 days were sacrificed and the microsurgery dissection protocol was conducted to remove the nerves and then observe the possible alterations at the transmission electron microscopy (TEM), by comparing a spinal nerve (sciatic nerve) with a cranial nerve (vestibulocochlear nerve/VIII Cranial nerve).
This could be the cause of aggressions, degenerations, atrophies, varying demyelination, polyneuritis and polyneuropathies of the peripheral nervous system, which affect the neurophysiological functions of these nerves.Malnutrition could change these neurophysiological mechanisms, as well as diabetesassociated malnutrition or non-associated, therefore it showed a new type of mixed peripheral neuropathy [5,6,7].With regard to the vestibulocochlear nerve (VIII cranial nerve) in large magnifications, the greatest evidence was axonal atrophy and cytoplasmic intumescent (Figures 1 and 2-5).
Figure 3 shows rats from the same broods, i.e. they present the same ages, but the rat is the Control (left side of the photo) and the mouse (right side of the photo) is malnourished and diabetic.Notice the great difference of sizes and the devastating macro effect of experimental diabetes- Source: Authors' original data.
associated malnutrition in rats, whose survival time is not long.With regard to the rats' sciatic nerve, some damage have been found in the diabetic nerve (Figure 4) compared to the control.In Figure 5, the effects of diabetes and malnutrition together are quite evident, causing greater damage to the sciatic nerve, analysis through the transmission electron microscopy (TEM).
Rats can develop similar diseases to those affecting human beings, like diabetes mellitus, commonly in an induced manner [8].Thus, it is possible to discover several complications of the disease, like neuropathy, and its associated factors that have great chances of similar development in humans [9].
Therefore, the following question can be raised: What are the factors associated with diabetic neuropathy in rats?
Diabetes mellitus is a public health issue that can cause serious consequences, such as retinopathy, kidney failure, hypertension, dyslipidemias, and neuropathy [10].This results in severe consequences for the subject, and therefore knowledge on healthdisease interfaces is essential, which will enable the adoption of an improved health practice [11].Source: Authors' original data.
Therefore, the aim of this study was to analyze the factors associated with diabetic neuropathy in rats.

Method
This is a systematic review with metanalysis based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol [12].Data collection followed some previously chosen election criteria, including (1) case-control articles or clinical trials, for they allow a better statistical analysis, (2) in any language, (3) full texts available, (4) between the years of 1995 to 2016.The eligibility criteria regarding year was chosen due to the large amount of research and publications on the theme since the year of 1995, when a study of great repercussion performed by ISHII (1995) was carried out, which approached growth factors similar to insulin in diabetic neuropathy pathogenesis, thus inciting other studies.Exclusion criteria were comprised of (1) articles presenting some bias risk or that (2) were inconclusive or (3) repeated studies.
The sources used for researched data were the Medical Literature Analysis and Retrieval System Online (MEDLINE/PubMed) and Scopus databases.The research was conducted using the keywords found in descriptors/MeSH: rats, neuropatias diabéticas/diabetic neuropathies, and risk factors, with the Boolean operator "AND".Phases regarding screening and eligibility for article inclusion in the systematic review and metanalysis were conducted for choosing the studies.Pairing was used for such purpose.Therefore, two authors worked independently by searching and selecting studies after their initial reading of titles and abstracts; in case of disagreement, a third reviewer was used to verify if the study was indeed eligible.Subsequently, a full analysis of each selected article was performed.
The analyzed variables regarding data extraction of the study were numbers of rats that developed or did not develop diabetic neuropathy and the factors associated with such characteristic.In studies that did not include some of the variables, the statistical process of multiple imputation was used to complete any possible existing voids.
Risks of bias of the selected studies were analyzed according to Downs and Black's (1998) [13] scale, which is comprised of the study quality analysis (10 items); ability to generalize study results (3 items); study bias (7 items); bias determination through the sample (6 items); and determination of random findings (1 item).Here, two reviewers also worked independently, and studies with scores lower than 7 points were excluded from the review.
The BioEstat 5.3 program was used for the statistical analysis and the Odds Ratio with a 95% interval confidence (CI) was calculated.It was possible to obtain a full analysis of the selected studies, thus enabling to find the chances of neuropathy development when it is associated with risk factors.The chi-square distribution was also calculated to verify the association between factors and neuropathy, and the Shannon's index was applied to check the behavior of the factors reported by the authors.

Results
Eighty-three references were found, of which 20 followed the criteria and were then included in the present review.The following Figure 6 includes the methodological steps followed to obtain the sample.Original pictures and data were included in the present review.
The next Table 1 shows the main factors associated with diabetic neuropathy development The following Figure 7 and Table 2 refers to the metasummary method of associated factors found in studies based on Shannon's index.
The next Figure 8 and Table 3 refers to the combined Odds Ratio of the analyzed studies.
Subsequently, the chi-square distribution was performed to check the association and hypothesis of nullity.(Figure 9)      Then, pieces of information regarding author and year, journal, purpose, and conclusion were extracted of the selected studies.(Table 4)

Discussion
A rodent has a chance of 2.9 times of developing diabetic neuropathy with the mentioned risk factors.The distribution of probability by means of the chi-square distribution showed a p-value of 0.0062 that associates the reported factors with the neuropathy development, thus representing the statistical significance of the analysis and rejecting the nullity hypothesis.
The period the animal has been presenting the disease is an important factor to be considered for the neuropathy development9.The more the animal remains with the disease, the higher are the chances of developing some kind of complication due to nervous damage, which can create other comorbidities when associated, such as the development of damaged vascularization [21].Morphometry is a method used not only in the daily laboratory routine, but also for investigational purposes, in order to collect data and analyze the results more objectively and accurately.
Vasconcelos 2011 [6] Int.J. Morphol To describe and compare ultrastructure alterations between a spinal nerve and a cranial nerve in rats with chronic induced diabetes.
Chronic STZ-induced diabetes in rats caused alterations in myelinated fibers and Schwann's cells that match with the classical signs and symptoms of diabetes.2013 [8] Diabetes Metab.To investigate the effect of G-CSF in peripheral nerves in the induced STZ in diabetic rats.
The present results show that G-CSF does not have damaging beneficial effects, but minimum ones regarding conservation of the peripheral nerve in diabetic rats.
Bakovic 2013 [9] Exp.Gerontol.To describe the quantitative alterations of intramyocardial nervous terminals in the hearts of diabetic rats of different ages.
Cardiac innervation suffers dynamic alterations both in Controls and in diabetic rats, with a significant time-dependent increase in the neuronal fiber density of diabetic rats.
Nithya; Ray 2014 [10] Int J Pharm Pharm Sci To study the effect of the six-week chronic treatment with Granisetron in neuropathy.
The chronic treatment of Granisetron significantly provides protection after the development of diabetic neuropathy.
Kara 2014 [11] Gynecol.Endocrinol To investigate ovariectomized rats, cerebellum cortex response to deficiency of estradiol and hyperglycemia.
Estradiol and insulin deficiency may affect brain cortex.

Metab. Syndr
To investigate the presence of peripheral neuropathy in spontaneously hypertensive rats (SHR) and SHR with chronicinduced diabetes.
Existence of neuropathy due to hypertension, which is among one of the most common risk factors of diabetic neuropathy.
To determine the inflammatory events in the CNS during Streptozotocin-induced diabetes.
Presence of Ang II-mediated cerebral inflammatory events in diabetes.
To test the hypothesis that the combination of hypertension, a risk factor for neuropathy in diabetic patients, with insulindeficiency diabetes, produces a more relevant model of peripheral neuropathy.
Combined rats with insulinopenia, hyperglycemia and hypertension provide a model for diabetic neuropathy.
To investigate the in vitro neurotoxicity and to block the in vivo duration in a genetic animal model of type 2 diabetes mellitus.
In vivo, subclinical neuropathy results in substantial extension of the blockage duration.
The triglycerides level is another important issue to be considered, because it is a factor that may compromise the vascular system and, as a consequence, the nervous system, resulting in peripheral nervous deficiency [10,18].In experiences done in animals and in the clinical practice with human beings, this is an important point to be considered based on this perspective [20].
Weight and blood pressure have also influenced the process of neuropathy development and they are usually associated in animals.These two factors may compromise the cardiovascular system and result in nervous damage, as well as in neuropathy development [2,14].When these factors are settled, many symptoms may appear, like pain and tingling, which are the main characteristics of the diabetes complication process [15,16].
These factors are associated with neuropathy development, even when the variables are adjusted for sex and age.Correlations higher than 50% are found in animals with these risk factors, even after excluding variables like sex and age.Thus, they show strong correlations between risk factors and the development of diabetic neuropathy [21].

Conclusion
Some factors, like nerve morphology, blood hypertension, oxidative stress, diabetes period, hypoglycemic effect, vascular complications, and insulin parameters or experimental situations with diabetes-induction using Streptozotocin ® represent the main risk factors for diabetic neuropathy development.
Such factors are very similar to those from humans, thus requiring a deeper analysis of the theme in a considerable human sample.There is still a lack of such theme in the scientific field, which should be filled.In animal models, like rats, we can make a good representation of these effects and of the Source: Elaborated by the authors damage to the nervous cellular structure, which can also be inferred to humans.Risk factors are important points to be considered in the health field, which should be analyzed in the most diversified circumstances for achieving a wider amplitude of the evidence level in such theme.Therefore, it is an important health epistemology and should be analyzed for a larger therapeutic direction in health.International Archives of Medicine is an open access journal publishing articles encompassing all aspects of medical science and clinical practice.IAM is considered a megajournal with independent sections on all areas of medicine.IAM is a really international journal with authors and board members from all around the world.The journal is widely indexed and classified Q2 in category Medicine.

Figure 1 :
Figure 1: Electron micrograph in cross-sectional cut of the VIII cranial nerve of a diabetic rat in a magnification of 5,000x that shows a fiber with axonal atrophy (Asterisk).

Figure 2 :
Figure 2: Electron micrograph in cross-sectional cut of the VIII cranial nerve of a diabetic rat in a magnification of 2,700x that shows, in smaller magnification, axonal atrophies and deformations/folds of the myelin sheaths (Head of arrows).

Figure 5 :
Figure 5: Electron micrograph in cross-sectionalcut of the sciatic nerve of a diabetic and malnourished Wistar rat in a great magnification of 14,000x that shows intumescent cytoplasm and Schwann cell nucleus, accumulation of granules, and an apparent demyelinated fiber (Asterisk).

Figure 4 :
Figure 4: Electron micrograph in cross-sectional cut of the sciatic nerve of a diabetic Wistar rat in a magnification of 6,700x that shows fibers with axonal atrophies (head of arrows).

Figure 3 :
Figure 3: Two rats (same age) of the same brood of the Wistar lineage: one is a Control and the other is malnourished and diabetic.

Table 1 .
Factors associated with the development of diabetic neuropathy.
Source: Elaborated by the authors

Table 4 .
Information from the included studies.
To explore alterations in microvascular permeability of the sciatic nerve, and if they are associated with the development of diabetic peripheral neuropathy.Permeability of sciatic nerves was associated with the development of neuropathy in the initial phase of diabetes in rats.
PNPD/CAPES Institutional, UFPE (Process number: 2311/2011).Nádia Nara Rolim Lima received a scholarship from PNPD/CAPES Institutional, PROPESQ/ UFPE of the POSNEURO Program.We thank the Bioterium of the Nutrition Department on behalf of the Neurocientist Professor Doctor Carlos Augusto Carvalho de Vasconcelos of the Laboratory of Experimental Nutrition and DIETETIC/LNED where is the chief coordinator.We would also like to thank the Neurosurgeon Professor Doctor Marcelo Moraes Valença for his supervision and technical provision, and also Laboratory of Immunopathology Keizo Asami (LIKA)/UFPE for the indispensable support on behalf of its director and main representative, Professor Doctor José Luiz de Lima Filho.we are completely grateful.we authors wish to thank the technician MR.Rafael Padilha for the excellent help and support needed.17.Lirk, P. et al.In Zucker diabetic fatty rats, subclinical diabetic neuropathy increases in vivo lidocaine block duration but not in vitro neurotoxicity.Reg.Anesth.Pain Med.37, 601-6 18. Kroin, J. S., Buvanendran, A., Tuman, K. J. & Kerns, J. M. Effect of acute versus continuous glycemic control on duration of local anesthetic sciatic nerve block in diabetic rats.Reg.Anesth.Pain Med.37, 595-600 19.Kim, H. et al.Bone marrow mononuclear cells have neurovascular tropism and improve diabetic neuropathy.Stem Cells 27, 1686-96 (2009).20.Zimering, M. B., Alder, J., Pan, Z. & Donnelly, R. J. Antiendothelial and anti-neuronal effects from auto-antibodies in subsets of adult diabetes having a cluster of microvascular complications.Diabetes Res.Clin.Pract.93, 95-105 (2011).21.Ishii, D. N. Implication of insulin-like growth factors in the pathogenesis of diabetic neuropathy.Brain Res Brain Res Rev 20, (1995).22. Nitta, A. et al.Diabetic neuropathies in brain are induced by deficiency of BDNF.Neurotoxicol Teratol 24, (2002).