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Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Abstract:  Diabetic neuropathy (DN) is the most common peripheral neuropathy and long-term complication of diabetes. In view of the pathological basis for the treatment of DN, it is important to prevent nerve degeneration. Most of the current treatment strategies are symptomatic therapies. In this study, we evaluated the effectiveness of magnesium-25, carrying porphyrin-fullerene nanoparticles, on diabetes-induced neuropathy. Previous studies have suggested that dorsal root ganglion (DRG) neurons comprise a specific target and may be responsible for the known complications of DN. Experimental DN was induced by intraperitoneal injection of streptozotocin (STZ) (45 mg/kg). Different forms of magnesium including 25Mg-PMC16, 24Mg-PMC16 and MgCl2 were administered intravenously in equal dose (0.5 LD50) at 48-hr intervals before STZ injection. Peripheral nerves were studied after 2 months of diabetes in groups using qualitative approaches, morphometric analysis of DRG neurons and motor function tests. We showed that STZ-induced DN caused morphological abnormalities in DRG neurons comprising changes in area, diameter and number of A and B cells as well as motor dysfunction in DN. Moreover, our findings indicated that administration of 25Mg-PMC16 as a magnetic form of Mg improved morphological abnormalities and motor dysfunctions significantly, whereas other forms of Mg were ineffective.

Diabetic neuropathy (DN) is the most common late complication of diabetes showing an increasing prevalence [1]. Sixty per cent of patients with diabetes show evidence of peripheral nerve disease [2]. Similar to other diabetes complications, DN has been ascribed to hyperglycaemia and subsequent metabolic abnormalities such as increased polyol pathway activity leading to the accumulation of sorbitol and fructose, imbalances in NADP/NAD+, auto-oxidation of glucose causing the formation of reactive oxygen species, advanced glycation end-products produced by non-enzymatic glycation of proteins, inappropriate activation of protein kinase C (PKC) and a deficit of neurotrophic supports [3]. The pathology of DN includes axonal atrophy, demyelination, loss of nerve fibres and decreased regeneration of nerve fibres [4]. Abnormalities of peripheral nerve function are typical late complications of diabetes. However, by applying streptozotocin (STZ) injections in rats, such a spectrum of disease characteristics can be seen much earlier, i.e. only several weeks after induction of diabetes [5].

Based on these observations, pharmacological therapies including aldose reductase inhibitors, antioxidant drugs, aminoguanidine and neurotrophic factors have been used in the treatment of DN [4]. Notwithstanding their importance, these treatments are solely symptomatic therapies, because peripheral nerve tissue damage cannot be reversed. Thus, early diagnosis of DN followed by a stringent pharmacological therapy may be effective to prevent the progression of DN, i.e. arrest the degenerative changes of nerve fibre pathology [4]. Oxidative stress mechanisms by induction of mitochondrial dysfunction, decrease in adenosine triphosphate (ATP) and neural death play important roles in peripheral neuropathy [6–8]. Our recent study also indicated the usefulness of PMC16 in DN in terms of improvement of oxidative stress and mitochondrial ATP production [9].

According to growing advances in the field of nanomedicine, we evaluated in this study the effects of magnesium-25 carrying porphyrin-fullerene nanoparticles that have the potential to prevent dorsal root ganglion (DRG) neuron degeneration and motor dysfunction symptomatically for DN. These nanoparticles are a novel pharmaceutical nano-tool based on the porphyrin-attached fullerene-C60 ‘ball’ (porphylleren-MC16 or PMC16) [6]. Regarding accumulation of this nanoparticle in neural mitochondria [6,10], we aimed to analyse the efficacy of PMC16 on morphological changes of DRG neurons and motor function recovery of DN rats in the present work. Therefore, the benefits of magnetic (25Mg-PMC16), non-magnetic (24Mg-PMC16) and ordinary (MgCl2) forms of Mg were compared in a model of experimental DN.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Subjects.  Male Wistar rats (200–250 g) obtained from the Tehran University of Medical Science, Faculty of Pharmacy, were used. All animal studies were performed according to the Ethical Committee for the use and care of laboratory animals of Tehran University of Medical Sciences. All efforts were made to minimise animal suffering and to reduce the number of animals used. Rats were placed in individual stainless steel cages, handled daily and given food and water ad libitum. A 12-hr light/dark cycle was maintained, and the experiments were performed during the light cycle.

Experiments.  All rats were divided into the following groups: control, DN, 24MgPMC16, 25MgPMC16 and MgCl2. The control group received normal saline as vehicles for STZ and different forms of Mg. The DN group received a single intraperitoneal (i.p.) injection of STZ (45 mg/kg). The 25Mg PMC16, 24Mg PMC16 and MgCl2 groups received two intravenous (i.v.) injections (48-hr interval) of 25Mg PMC16, 24Mg PMC16 and MgCl2 alone or followed by an i.p. injection of STZ after 2 days. According to our previous work, pharmacokinetic information of nanoparticle including LD50 and the literature [6,9], various doses of nanoparticles were evaluated. At the beginning, we used a concentration <0.5 LD50, but no proper responses were obtained and finally the suitable dose of 0.5 LD50 was selected. After finding this dose, the effects of 25Mg PMC16 as a magnetic isotope were compared with non-magnetic isotope (24Mg PMC16) and ordinary form of magnesium (MgCl2) in all parts of the experiments. Different forms of magnesium were administered with similar dose (0.5 LD50 = 448 mg/kg for 25Mg PMC16 and 24Mg PMC16 and 88 mg/kg for MgCl2).

Diabetic neuropathy in type 1 diabetic streptozotocin (STZ) models.  Diabetes mellitus type 1 was induced by the i.p. injection of dissolved STZ (45 mg/kg) in normal saline with a pH of 7. Animals were fasted for 12 hr and then injected with a STZ solution. Within 1 week of injection, the animals became hyperglycaemic with blood glucose levels between 250 and 500 mg/dl. Two months after confirmation of hyperglycaemia, DN occurred [11].

Sample preparations.  Blood and tissue samples were obtained by anaesthetising the animals with an intraperitoneal (i.p.) injection of ketamine (80 mg/kg) and xylazine (20 mg/kg). Then, the animals were killed, and 1–3 ml heart blood was collected into heparinised syringe for the evaluation of stress oxidative parameters.

For tissue preparation, the spinal cord and para spinal tissue from the second cervical to the second lumbar spine region were removed from the anaesthetised animals. To be specific, the spines were broken and all vertebrates were separated. After removing cervical spine lamina, the spinal ganglia were exposed and the sixth cervical (C6) DRG on each side was removed. DRG neurons were fixed by 10% formalin for the morphological study [12].

Evaluation of oxidative stress biomarkers.  Total antioxidant capacity, sulfhydryl level of thiol groups and lipid peroxidation were determined in plasma as described in our previous study [9].

Histological preparation.  DRG neurons were dehydrated in an ascending series of ethanol and then passed to xylol for removal of alcohol. Then, the samples were embedded in paraffin and sectioned by a microtome setting of 40 μm. With a random starting point within the first five sections, every fifth section was collected for HE staining. The HE-stained sections were used for microscopic analysis.

Morphometry of DRG neurons.  DRG cells were defined as either A or B cells. The nucleus of A cells has one large intensely stained central nucleus, and the cytoplasm appears granular. In the periphery of the cytoplasm of some of the cells, an unstained area can be seen.

B cells are characterised by a nucleus containing multiple peripherally located nuclei. The cytoplasm is more homogeneous and more intensely stained than that of A cells. In some cells, the central cytoplasm is only lightly stained, while the outer parts are intensely stained [5]. We used an Olympus microscope (LX71, Japan) to determine the number, diameter and area of A and B cells in all groups. Then, we used an image evaluation programme (Optika, Italy) for stereological studies.

Measurement of motor function.  Motor function studies were evaluated by open-field activity tests. The rats were placed in an open-field box where activity, discrete movements such as movement time (sec.), travelled distance (cm) and velocity (cm/sec.) were recorded by a camera on the top of the box for 15 min. [13,14]. The EthoVision tracking system (Noldus Information Technology, Wageningen, the Netherlands) was used to evaluate motor function by measuring time, distance and speed of animal movement.

Materials.  MgCl2, diethyl ether, xylol, formalin, hematoxylin-eosin, ketamine, xylazin, erythrosine and alcohol were from Merck (Frankfurt, Germany), STZ from Pharmacia & Upjohn Inc. (Kalamazoo, New Jersey, USA), 25Mg PMC16 and 24Mg PMC16 from Semenov Institute, Russian Academy of Sciences, Moscow.

Statistical analysis.  All values were expressed as means ± S.E.M. Data were statistically analysed by anova followed by Newman–Keuls post hoc test. p-values <0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Qualitative effects of different types of Mg on DRG neurons observations.

We observed in HE-stained DRG neurons a prominent vacuole formation and under-representation of the largest diameter clear neurons (type A) and possibly an over-representation of small, more basophilic neurons in DN animals (type B) compared with controls. After 25Mg-PMC16 administration, we observed notable improvement changes in DRG neurons compared with the DN group, but we did not find any observable difference in the 24Mg PMC16 or MgCl2 groups in comparison with DN animals (fig. 1A–E).

image

Figure 1.  HE-stained dorsal root ganglion neurons. Control (NS) (A), diabetic neuropathy (DN) (STZ) (B), 25Mg-PMC16 + STZ (C), 24Mg-PMC16 + STZ (D) and MgCl2 + STZ (E) groups. DN neurons tend to be small and more basophilic and with more and larger vacuoles. Also, we observed a decrease in A cell size and conversion of A cells into B cells. Infusion of 25Mg-PMC16 (C) abolished these effects. Scale bar 50 μm (400×).

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Analysis of morphometric parameters of A and B neurons in DN and 25Mg-PMC16 nanoparticle-treated animals.

The number of small neurons (B cells) considerably exceeded that of large neurons (A cells) in DN as compared to controls (***p < 0.001, fig. 2A,B). There was a significant increase in the number of large cells (***p < 0.001) in the 25MgPMC16-treated group in comparison with DN rats (fig. 2A). 25Mg PMC16 also counteracted the DN-induced changes in B cell numbers compared with the DN animals (**p < 0.01, fig. 2B). Area (μm2) and diameter (μm) of large neurons as well as the area of small neurons were significantly reduced in DN compared with control animals (***p < 0.001 and *p < 0.05, respectively, table 1). The application of the magnetic isotope of Mg-nanoparticles was able to prevent these DN-mediated changes. However, a significant increase in the area and diameter of large cells (#p < 0.001) was observed after 25MgPMC16 treatment. No effects were seen using 24Mg PMC16 or MgCl2 (table 1).

image

Figure 2.  Protective effects of magnesium types on the number of large (A) and small (B) cells in diabetic neuropathy (DN) rats. The number of A cells considerably decreased in DN as compared to controls (***p < 0.001). DN induced significant increase in B cells in comparison with control rats (***p < 0.001). 25Mg-PMC16 counteracted the changes in A and B cell numbers compared with the DN group (***p < 0.001, **p < 0.01). No effects were seen by applying 24Mg-PMC16 or MgCl2. Data represent means ± S.E.M. of six animals.

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Table 1.    Protective effects of magnesium types on dorsal root ganglion neurons size in diabetic neuropathy (DN) rats.
 Diameter (μm)Area (μm2)
GroupsA cellsB cellsA cellsB cells
  1. DN decreased the diameter and area of A cells (***p < 0.001), as well as the area of B cells (*p < 0.05) compared with their respective control groups. 25MgPMC16 administration to DN groups showed a significant restoration of A cell diameter and area (#p < 0.001) compared with the DN group. The 24MgPMC16- and MgCl2-treated DN animals showed the same statistical changes as the DN group compared with controls. Values represent the means ± S.E.M. of six animals.

Control47.13 ± 1.8332.30 ± 0.911326 ± 78.03836.8 ± 42.1
DN32.82 ± 1.50***26.76 ± 1.31775.3 ± 75.48***601.0 ± 50.47*
25MgPMC1647.28 ± 1.83#31.68 ± 2.211308 ± 89.43#768.5 ± 45.55
24MgPMC1633.27 ± 2.28***27.31 ± 1.53794.9 ± 83.3***599.5 ± 69.15*
MgCl232.40 ± 2.20***27.25 ± 1.47695.7 ± 84.74***603.3 ± 60.80*

Measurements of oxidative stress parameters.

Similar to our previous experiment [9], an increase in total antioxidant capacity and total sulfhydryl molecules and decrease in lipid peroxidation were observed in 25MgPMC16-treated animals in comparison with the DN group. Administration of 24Mg PMC16 and MgCl2 did not cause any significant alterations in oxidative stress biomarkers (data not shown).

Evaluation of motor function deficiency.

Motor function was measured immediately before STZ injection (day 0) and on days 7 and 60 after STZ administration. Two months after induction of diabetes, a clear deficiency in motor function was present in DN rats. The movement duration (sec.) (fig. 3A, ***p < 0.001), distance moved (cm) (fig. 3B, ***p < 0.001) and velocity (cm/s) (fig. 3C, **p < 0.01) were significantly lower in DN than in control rats. Infusion of 25Mg-PMC16 nanoparticles improved motor functions and reversed slowing of movement time (sec.), as well as distance moved (cm) and velocity (ap < 0.01) significantly in comparison with DN rats. There was no significant difference between 25Mg-PMC16-treated animals and control groups (fig. 3A,B,C). The two other types of magnesium did not reveal any significant changes (fig. 3A,B,C). Single administration of each form of magnesium did not affect motor function significantly.

image

Figure 3.  Protective effects of magnesium types on motor activity movement duration (sec.) (A), distance moved (cm) (B) and velocity (cm/sec.) (C) at 7 and 60 days after STZ (45 mg/kg) administration. The motor function was also measured immediately before STZ injection (day 0). Administration of 25Mg-PMC16 caused significant increase in movement duration (sec.) (A), distance moved (cm) (B) and velocity (cm/sec.) (C) (ap < 0.01) compared with the diabetic neuropathy group, whereas the infusion of 24Mg- PMC16 or MgCl2 had no effect. Data represent the means ± S.E.M. of six animals.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The major findings of this work were as follows: (i) 25Mg-PMC16 prevented degeneration of DRG neurons in DN rats; (ii) This type of Mg nanoparticle improved motor function deficiencies in the DN group; and (iii) Other types of Mg did not compensate or ameliorate DN effects.

In the present study, we used amphiphilic and membranotropic nanoparticles that contain ferroporphyrin and fullerene in their structure to analyse their potency to diminish cell destructive effects seen under DN-like conditions in rats. These nanoparticles serve as nanocation particles both in vitro and in vivo and are putatively beneficial in terms of regulating ‘smart release’ of magnesium in hypoxia-induced acidosis in DN. They activate oxidative phosphorylation pathways and increase ATP level. They are safe to use because non-allergic and anti-inflammation properties of most fullerene derivatives ever tested as well as their generally high level of ‘biocompatibility’ make PMC16 suitable for in vivo safety and pharmacological activity studies [6]. Some data also showed that porphyrin-modified negatively charged fullerenes were completely safe products with no tendency to interact with immunological responses of any sort [6]. In addition, the hepatic metabolic attack occurs mainly on the porphyrin domain of PMC16, which causes the formation of haem precursor with neutral biological properties [6]. These effects, along with high rate of renal elimination, lead to consider PMC16 particles perfectly safe for chronic administration as well. These are the reasons why we observed no notable adverse effects and confirm additional support for safety potential of the PMC16 nanoparticles.

As generally accepted, 2,3-DPG (2,3-diphosphoglycerate) resembles a biological indicator of tissue hypoxia and DN [15]. A decrease in the red blood cell 2,3-DPG concentration, which is one of the factors contributing to tissue hypoxia induced by DN, has been reported previously [9]. The decrease in erythrocyte 2,3-DPG concentration in DN rats might be due to hyperglycaemia-induced metabolic deficits that would lead to erythrocyte dysfunction. Erythrocytes with normal functions can increase 2,3-DPG in the hypoxic condition, but abnormal erythrocytes in DN show an increased oxygen affinity and decreased oxygen release [16]. Recently, we have shown that 25Mg-PMC16 nanoparticles improve 2,3-DPG in DN [9].

Progressive loss of sensory fibres in peripheral nerves is a characteristic phenomenon of DN and is accompanied by degeneration and loss of parent DRG neurons. Hyperglycaemia and subsequent metabolic abnormalities such as increased polyol activity, nerve hypoxaemia and oxidative stress have been suggested as the main causes of DN [2,7,8]. Motor and sensory conduction abnormalities are present in chronic DN. Because motor conduction defects emerge early during DN and progress in a period of time [17], we evaluated motor functions on days 0, 7 and 60 after STZ administration.

In general, neurons from diabetic rats tend to be smaller, reveal a stronger basophilic staining attitude and have more and larger vacuoles [18]. There is a selective atrophy of the largest DRG neurons in DN. Total count of A and B cells are almost equal in control and under DN conditions, but the ratio of large to small neurons is reduced in diabetic rats. Nerve conduction studies showed reduced conduction velocities in DN [18]. These findings were confirmed by our results. It is conceivable that an impaired synthesis of neuroskeletal proteins may play a role. The vacuolar changes are likely to be caused by an impaired neuronal energy metabolism and perturbed sodium handling possibly via modified Na+/K+-ATPase activity and/or decreased DRG blood flow in DN [2].

Conduction velocity is directly related to the size of DRG neurons. In DN models, however, atrophy or loss of DRG neurons appears much later than do changes in conduction velocity [19]. It is believed that a slowing of conduction may be the cause of metabolic changes. These changes are associated with impaired mitochondrial generation of high-energy metabolites such as ATP [19].

A decreased mitochondrial membrane potential induced by diabetes can also cause a decrease in ATP synthesis. Impairment of the bioenergetic system of sensory neurons alters cellular functions [20] that results in atrophy of neurons and consequently in a deficiency of motor function. Motor abnormality which is because of the mitochondrial dysfunction and accompanied by an accumulation of oxygen radicals indirectly supports this ‘oxidative hypothesis’ [21].

As we have shown in previous studies, the decrease in 2,3-DPG, hypoxia and a subsequent acidosis in DN are conditions that activate nanoparticles [9]. Under such conditions, 25Mg-PMC16 can increase ATP and thereby affect phosphorylation and oxidation of substrates. This can ameliorate neuronal atrophy and motor function deficiency. A neuroprotective strategy involves mitigating oxidative stress, which is believed to be a neuropathological key process that contributes to degenerative disorders [22]. Fullerenes and metalloporphyrins are also known as neuroprotective agents [21,23]. The neuroprotective activity of fullerenes is based on their capability to react with oxygen radical species such as superoxide (O2˙) and hydroxyl (˙OH) radicals [23]. It was shown that metalloporphyrins such as ferroporphyrins possess cell protective effects which are related to their ability to detoxify superoxide, H2O2 and peroxynitrite [21].

Our findings indicate that 25Mg-PMC16, by affecting stress oxidative biomarkers such as total antioxidant capacity, sulfhydryl molecules and lipid peroxidation, induce antioxidant effects (data not shown). These data confirm the results of our previous work [9]. Considering the antioxidant properties of both magnesium and fullerene derivatives, it is thus reasonable to deduce that 25Mg-PMC16, by significant elevation of intra-neuronal Mg and positive role of fullerene structure, improves DN-mediated damage of DRG neurons and motor dysfunction in DN.

The porphyrin receptors on the mitochondrial membrane are tissue selective sites for interaction with the porphyrin domain of PMC16. The water-soluble activity of C60-fullerene derivatives provides selective ability for accumulation of PMC16 inside mammalian mitochondria [6]. Regarding the reports indicating that DRG neurons have large mitochondria with a highly oxidative metabolism which makes these much susceptible to oxidative injury [10], these nanoparticles can be navigated towards the DRG neurons in a suitably targeted delivery manner. However, the mitochondrial intake, PMC16 pharmacokinetics, particle size and the porphyrin domain–receptor recognition are involved in penetration and accumulation of nanoparticles in DRG neurons.

Another finding of this study is the days long-lasting protective effects of 25Mg-PMC16. The most plausible explanation for this effect is a presence of the PMC16 high-affinity receptors in mitochondrial membrane and activation of a signalling pathway by selective drug–receptor interaction. A protein signalling cascade is known for a number of porphyrin derivatives [6,24–26]. Moreover, the resistance of PMC16 to hepatic metabolism and increase of its half-life in hypoxia conditions [6] are other possibilities for accumulation and retaining of PMC16 in DN-DRG neurons for a longer period of time.

It should be mentioned that we observed beneficial effects only with 25Mg-PMC16 and not with other forms such as 24Mg-PMC16 and MgCl2. Some reports demonstrated that the separate and targeted PMC16 delivery of magnetic and non-magnetic isotopes provide a high level of ATP production once the magnetic isotope has been applied [6]. On the other hand 25Mg-PMC16 is unable to release Mg efficiently in plasma with normal pH of 7.4 but when intracellular pH will alter to acidic level by cellular damage or hypoxic condition, the magnetic form of nanoparticle can release Mg as much as causing a significant increase in Mg inside DRG cells. Because we used equal doses of three forms of Mg in our study, the first reason to be concluded is that magnetic magnesium by better reaching and accumulation in neuronal cells shows a protective effect much better than other forms of Mg. However, the positive effects of metaloporphyrin and fullerene should not be excluded.

Taken together, our study demonstrates that multiple cellular and molecular pathways are responsible for DN-mediated damages in DRG neurons which are associated with neuronal degeneration, atrophy and impaired motor function. The potency of 25Mg PMC16 to abolish the observed toxic effects may open novel therapeutic aspects for the treatment of diabetic symptoms associated with neuronal destruction.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by funds from Tehran University of Medical Sciences.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References