Neuroprotective Effect of Cerebrolysin on Diabetic Neuropathy: A Study on Male Rats

Diabetes mellitus is a disorder recognized with increase of blood sugar level. Impaired insulin release or failure to respond to insulin or both is the cause of this disorder. Chronic hyperglycemia leads to the dysfunction of several organs especially eye, kidney, heart and vessels [1]. Peripheral neuropathy is one of the common complications of diabetes which in turn increases the risk of other diabetes complications such as foot ulcers and amputation [1]. Almost more than half of diabetic patients suffer from different forms of neuropathy after passing 1-2 decades of their disease [2,3]. In animal models of streptozotocin-induced diabetes, this time has decreased to at least two weeks [4]. Peripheral diabetic neuropathy is the result of several factors [5-7] and its probable mechanisms include glycosylation of neural proteins, microangiopathy, neuronal antibodies and ischemia resulted from basement membrane thickening of the vasa nervorum. Abnormalities of polyol pathway and defects of protein kinase C metabolism which cause nerve demyelinating have also been described in diabetic peripheral neuropathy [6]. Based on these mechanisms of injury, various prevention and treatment strategies have been already suggested and are under investigation. For instance, the effects of several antioxidants such as vitamin E [8], melatonin [9] and date extract [10], fatty acid contained diets like Omega 3 [11], aldosereductase inhibitors [12] and also statins compounds such as atorvastatin [13] have been investigated, but none of them have already been approved by FDA. At present, there is no definite treatment for this complication.


Introduction
Diabetes mellitus is a disorder recognized with increase of blood sugar level. Impaired insulin release or failure to respond to insulin or both is the cause of this disorder. Chronic hyperglycemia leads to the dysfunction of several organs especially eye, kidney, heart and vessels [1]. Peripheral neuropathy is one of the common complications of diabetes which in turn increases the risk of other diabetes complications such as foot ulcers and amputation [1]. Almost more than half of diabetic patients suffer from different forms of neuropathy after passing 1-2 decades of their disease [2,3]. In animal models of streptozotocin-induced diabetes, this time has decreased to at least two weeks [4]. Peripheral diabetic neuropathy is the result of several factors [5][6][7] and its probable mechanisms include glycosylation of neural proteins, microangiopathy, neuronal antibodies and ischemia resulted from basement membrane thickening of the vasa nervorum. Abnormalities of polyol pathway and defects of protein kinase C metabolism which cause nerve demyelinating have also been described in diabetic peripheral neuropathy [6]. Based on these mechanisms of injury, various prevention and treatment strategies have been already suggested and are under investigation. For instance, the effects of several antioxidants such as vitamin E [8], melatonin [9] and date extract [10], fatty acid contained diets like Omega 3 [11], aldosereductase inhibitors [12] and also statins compounds such as atorvastatin [13] have been investigated, but none of them have already been approved by FDA. At present, there is no definite treatment for this complication.
Cerebrolysin is a neuropeptide anti-inflammatory mixture isolated from pig brain tissue [14]. It is a neurotrophic peptidregic mixture resulted from enzymatic breakdown of free-lipid porcine brain proteins. It contained 25% low molecular weight peptides (<10 KDA) and 75% free amino acids depending on free nitrogen content [15]. Cerebrolysin contains relatively high concentrations of magnesium, potassium, phosphorus, selenium [16] and also other elements [17,18].
Cerebrolysin was first used in 1973 [19] as a hydrolysate in patients with cerebral arteriosclerosis. It has been suggested for several types of nerve degeneration disorders [20][21][22], organic mental disorders [21], Multiple sclerosis [22], anti-aging [23] and ischemic encephalopathy [24]. This medicine has also been applied in the treatment of pediatrics cerebral paralysis, elderly patients and some other conditions [25]. It has been recognized in a comparative study that antioxidant properties of cerebrolysin is approximately 300 times less than that of trolox (vitamin E) [26].
In regard to the positive effects of cerebrolysin on neurotic disorders reported in previous studies, the present study was designed to investigate cerebrolysin as an effective mixture in the process of ischemia and improvement of diabetic neuropathy.

Animals
In the present study, male NMRI rats weighing 250 ± 25 gr kept in the animal house of Kerman Neuroscience Research Center were used. Animals were kept at 25 ± 1°C, 12/12 h light/darkcycle and free access to the same food and water. The study protocol was approved by the animal ethics committee of this institution (Code:EC/KNRC/88-15).

Diabetes induction
Rats were divided into four groups: First group consisted of control animals. The second, third and fourth groups consisted of diabetic animals (at least 8 rats in every group).
In order to induce diabetes, streptozotocin purchased from Sigma Company(65 mg/kg in 100 mmol/L sodium citrate buffer, pH4.5) was intraperitoneally injected into at least 24 rats [27]. One week after STZ injection, animals with fasting blood sugar higher than 200 mg/ dl were selected for experiments. Six weeks after the injection of STZ and appearance of neuropathy in diabetic animals [27,28], First group (Control): received nothing, second group (Diabetic): received nothing, third group (Vehicle): received 5 ml/kg day -1 saline intraperitoneally for 2 weeks and fourth group (Experimental): received 5 ml/kgday -1 cerebrolysinintraperitoneally for two weeks. This dose of cerebrolysin has been identified as neuroprotective inducing dose in nerve injuries [29,30].

Tail flick test
Tail flick is one of the standard tests for measuring the rate of analgesia. In this test, thermal light with the intensity of 5 was directed on the distal part of the animal's tail by Tail flick instrument (made by Spanish Lsi LETICA, model LE7406) and tail flick latency was measured. In order to prevent tissue damage, light was directed for a maximum time of 10 seconds. For each animal, tail flick latency was measured three times with 5 minutes intervals and mean of them was reported as tail flick latency [31].

Open field test
Open field test was used in order to survey the effect of diabetic neuropathy on exploratory behavior of diabetic rats and the probable protective effect of Cerebrolysin. Exploratory behavior was investigated by a video tracking system (TSE) in a 45*45*45cm box. At the end of 6th week after diabetes induction, animals were placed in the center of the arena and their exploratory behavior including horizontal, central and peripheral paved distances were measured for 5 minutes. The duration of staying in the center and peripheral parts as well as the velocity of movement were investigated [32].

Electrophysiological evaluation
Six weeks after the initiation of hyperglycemia, animals were anesthetized with intraperitoneal injection of 50/20 mg/kg ketamine+ xylazine solution. The environment temperature was kept at 25 ± 1°C during all phases of experiments. After shaving back of animal's leg, a small incision was made in the right sciatic notch and ankle. Then, using bi-polar electrodes, the proximal part of sciatic nerve at the sciatic notch and the distal part at the ankle were stimulated and motor neuron conduct velocity (MNCV) of sciatic-tibial motor nerve was recorded (powerlab/ML856;AD Instruments, Sydney, NSW, Australia). Immediately after stimulation, action potential of the first interosseous muscle of back paw was recorded by mono-polar electrodes. The obtained records are biphasic responses with one primary m wave appeared due to the stimulation of motor fibers. Motor nerve conduct velocity (m/s) is calculated through dividing the distance between two stimulated points (mm) by the time difference of two stimulations [10,33].

Histopathological evaluation
After the experiments related to NCV, animals were anesthetized with 400 mg/kg chloral hydrate and following cardiac perfusion by using saline and bouin's fixative [34]. Then a section of sciatic nerve (1 cm) was removed and kept in bouin's fixative. Forty eight hours after remaining in fixative, it underwent tissue processing phases and was embedded in paraffin. Then 4 µm sections were prepared and stained with hematoxylin-eosin for surveying by light microscope(Motic Images China e-kup Co., Ltd)(x400) [35].
In this evaluation, axons of nerves in sciatic transversesection were investigated in regard to edema andaxoplasm state (demyelination and remyelination) [36].

Statistical analysis
Parametric paired t-test was used for comparison of coupled primary and secondary variables and in order to compare quantitative variables among groups ANOVA was applied. Tukey test was used in the case of significant difference and in the case of rejection of null hypothesis, non-parametric Kruskal Wallis was applied.

Metabolic parameters
In all diabetic groups, mean plasma concentration in the 8 th week was 280% more than that in the control group. All diabetic rats showed high blood sugar and weight gain disorder in the 8 th week after STZ injection. As it has been presented in Table1, weight of diabetic animals compared to the non-diabetic control group had significantly decreased in the 8 th week ( Table 1).

The effect of cerebrolysin on tail flick test
Diabetic neuropathy caused significant increase in reaction to pain and tail flick latency time in diabetic, vehicle and cerebrolysin groups compared to the control group, but diabetic and vehicle groups showed no significant difference with cerebrolysin-received group in this regard ( Figure 1).

Nerve Conduct Velocity
In regard to mean NCV, vehicle and diabetic groups showed significant difference with the control group (p=0.000 and p=0.000 respectively), and also they had significant difference with cerebrolysin-  group (p=0.012 and p=0.024 respectively). While There are no significance effects between cerebrolysin-treated and control groups (p=0.107). This finding shows significant efficacy of cerebrolysin in improving nerve function in the cerebrolysin-received group compared to vehicle and diabetic groups ( Figure 3).

The effect of cerebrolysin on morphological alterations of nerve myelin
Microscopic investigations showed normal structure and morphology of myelin in the control group, but in the diabetic and vehicle groups, edema and myelin sheath splitting were observed. Increase in the number of fibers with abnormal myelin was observed in the diabetic and vehicle groups. Treatment with cerebrolysin could prevent abnormal cases in a wide extent. Data related to the myelin and axon diameters of nerve fibers in the vehicle and diabetic groups showed significant decrease in comparison to the control group. Cerebrolysin administration caused significant improvement in myelin and axon diameter reduction among diabetic rats in two weeks ( Table  2 and Figure 4).

Discussion
In several in vivo and in vitro studies, neuroprotective and neurotrophic effects of cerebrolysin have been reported. Cerebrolysin has caused improvement in cell oxidative stress during cerebral ischemia in animals.
Considering the identified properties of cerebrolysin as a neuropeptide anti-inflammatory mixture [14] and also previous related studies, the probable mechanism of cerebrolysin effect on diabetic neuropathy can be explained as follow.
Since ischemia is associated with decrease of blood flow followed by oxygen and food shortage and consequently stopping of energy production in tissue vessels, it has a significant role in producing and extension of pathologic changes in various neuropathies including peripheral neuropathies especially in sciatic nerve. These pathologic alterations in nerves are related to the degeneration of fibers and edema. In ischemia phase, blood flow stopping and oxygen shortage result in anaerobic metabolism, energy loss, ATP decrease and accumulation of hypoxanthine in ischemic cells. Lack of energy affects ATPase ion pump of cell membrane and causes sodium, calcium and water accumulation and consequently cell edema [37].
Gusev et al. [38], treated 30 patients with severe ischemic strokes by administering 10, 20 and 30 mg/day cerebrolysin for 10 days and have reported improvement in patients with moderate disease in comparison to their control group. Indeed, cerebrolysin improves motor activities and EEG signals in rats with moderate ischemia in anterior portion of brain. Also cerebrolysin has high neurotrophic property due to having very useful compounds such as 25% low density proteins (KDA<10), 75% free amino acids [15], high concentrations of magnesium, potassium, phosphor and selenium [16] and also some other elements [18,38]. This medicine, through providing the nerve cell with these elements, helps both cell metabolism process and remyelination.
Neuroplasticity involves the activation of existing but silent connections, synaptogenesis, dendritic arborization and new nervous cell production [17]. Neuroplasticity can be enhanced by administration of neurotrophic factors [39]. Due to its unique composition consisting of active fragments of neurotrophic factors, Cerebrolysin is able to confer neuroprotection and to stimulate neuroplasticity, thereby enhancing the neurorecovery process [40,41].
The other mechanism for explaining cerebrolysin effect is its antioxidant property [26,42]. In the process of diabetic neuropathy, nerve cells and vessels' membranes are not dependent to insulin for transferring glucose and in diabetes disorder great amount of glucose enter cells. In nerve cells, glucose changes to sorbitol by aldose reductase enzyme and sorbitol accumulation increases free radicals such as hydroxyl-super oxide and hydrogen peroxide and eventually causes cell damage. Based on this mechanism of injury, different prevention and treatment approaches are under investigation [43,44]. As it was mentioned in the introduction, anti-oxidant property of cerebrolysin is 300 times less compared to vitamin E [26]. Therefore, it seems that cerebrolysin with its minor anti-oxidant property could remove free radicals to some extent and caused improvement of diabetic neuropathy.
Diabetic neuropathy in its early stages is associated with increase of nerve fiber activity and disorder of normal sensitivity of peripheral nervous system to injuries and painful stimulators resulted from diabetic hyperalgesia [33,45]. However, after passing early stage the sensitivity of peripheral nerves decreases and caused various range of analgesia.
According to the obtained results in the present study, cerebrolysin (5 ml/kg/day, ip) can exert positive effects within two weeks in the treatment and decreasing the physiological symptoms of diabetic neuropathy in male rats. In the present study, response time to thermal pain in tail flick test showed significant increase in the diabetic group in comparison to the control group that is due to diabetic analgesia. Cerebrolysin could not significantly reduce this analgesia [46].   In open field test, it was seen that although cerebrolysin cannot exert significant improvement in behavioral variables in comparison to diabetic controls, in some extent it can improve (even though nonsignificantly) diabetic neuropathy.

MSD (μm) AD (μm) MMFD (μm)
Mean NCV in the diabetic group showed 50% reduction in comparison to the control group that shows high neuropathy percentage in diabetic rats. Indeed, it was observed that intraperitoneal injection of cerebrolysin can significantly cause improvement of NCV in neuropathy-induced male rats.
The presence of abnormal fibers in sciatic nerve that showed degenerative changes of axon and myelin splitting was one of the other symptoms of STZ-induced diabetic rats. In fact, one of the main reasons of nerve activity reduction in the process of diabetic neuropathy disorder, is morphological changes occurred due to nerve metabolic disturbances. In the present study, we observed the efficacy of cerebrolysin in improving morphological injuries of sciatic nerve myelin in rats with diabetic neuropathy. Morphological observations showed remyelination after two weeks of treatment with cerebrolysin. Axon diameter (AD), myelin sheath diameter (MSD) and mean myelinated fiber diameter (MMFD) showed absence of any significant difference between cerebrolysin-received rats and controls; however, means of all these indices were higher in the control group as compared with the cerebrolysin-received group. Means of AD, MSD and MMFD in vehicle and diabetic groups had significant decrease in comparison to the control group. This finding shows the efficacy of cerebrolysin in the improvement of degenerated axons of nerve fibers or remyelination in STZ-induced diabetic rats.
As we mentioned above, our study showed that cerebrolysin (5 ml/ kg/day, ip) can exert positive effects within two weeks in the treatment of diabetic neuropathy in male rats. We also expect to have more significant effect of cerebrolysin at a different dose level or duration. Therefore complementary studies should be done to reveal the optimum dose and duration.

Conclusion
It was observed in the present study that intraperitoneal injection of cerebrolysin is effective in the treatment of diabetic neuropathy and can improve the function of peripheral nerves.