Pharmacological, morphological and behavioral analysis of motor impairment in experimentally vitamin C deficient guinea pigs

Análise farmacológica, morfológica e comportamental do comprometimento motor em cobaios com deficiência de vitamina C induzida experimentalmente

Abstracts

The scurvy shows an inflammatory disease and gingival bleeding. Nevertheless, in an animal model for guinea pigs, described by Den Hartog Jager in 1985, scurvy was associated with a motor neuron disease with demyelinization of the pyramidal tract, provoking neurogenic atrophy of muscles. Aiming at searching the protective role of vitamin C in nervous system, a pharmacological, morphological and behavioral study was conducted. Three experimental groups were used: A100, animals receiving 100 mg/ vitamin C/ day; A5.0, animals receiving 5.0 mg/vitamin C/ day; and A0, animals without vitamin C. We analyzed the weight gain, muscular diameter and behavioral tests. In all tests examined, we found significant differences between the supplemented groups in comparison with scorbutic group (p<0.05). Thereafter, the animals were killed for histopathology of gastrocnemius muscle, spinal cord and tooth tissues. In addition, a morphometric study of periodontal thickness and alpha-motor neuron cell body diameter were done. The vitamin C-diet free regimen seemed to induce a disruption in spinal cord morphology, involving the lower motor neuron, as confirmed by a significant reduction in neuron perycaria diameter and muscular atrophy, complicated by increased nutritional deficit.

vitamin C; ascorbic acid; motor neuron disease; guinea pig; motor behavior

O escorbuto se caracteriza por doença inflamatória e sangramento gengival. Contudo, num modelo animal em cobaios, descrito por Den Hartog Jager em 1985, o estado escorbútico foi associado à doença do neurônio motor com desmielinização do trato piramidal, provocando atrofia neurogênica dos músculos. Objetivando investigar o papel protetor da vitamina C no sistema nervoso, um estudo farmacológico, morfológico e comportamental foi conduzido. Três grupos experimentais foram usados: A100, animais recebendo 100 mg/vitamina C/ dia; A5,0, animais recebendo 5,0 mg/ vitamina C/ dia; e A0, animais sem vitamina C. Nós avaliamos o ganho de peso, diâmetro muscular e realizamos testes comportamentais. Em todos os testes examinados, detectamos diferenças significantes dos grupos suplementados em relação ao grupo escorbútico (p<0,05). Os animais foram sacrificados para histopatologia do músculo gastrocnemius, medula espinhal e tecidos dentários. Também foi realizado estudo morfométrico da espessura do periodonto e do diâmetro dos corpos celulares dos neurônios motores alfa. A carência de vitamina C parece comprometer a morfologia da medula espinhal envolvendo o neurônio motor inferior, confirmada pela redução significativa do diâmetro dos pericárions e atrofia muscular, complicada pelo aumento do déficit nutricional.

vitamina C; ácido ascórbico; doença do neurônio motor; cobaio; comportamento motor

Pharmacological, morphological and behavioral analysis of motor impairment in experimentally vitamin C deficient guinea pigs

Análise farmacológica, morfológica e comportamental do comprometimento motor em cobaios com deficiência de vitamina C induzida experimentalmente

Reinaldo Barreto Oriá^I; Carlos Maurício de Castro Costa^II; Terezinha de Jesus Teixeira Santos^III; Carlos Meton de Alencar G. Vieira^IV

Laboratory of Experimental Neurology and Neurophysiology, Department of Physiology (DFF) and Pharmacology, Federal University of Ceará (UFC) Fortaleza CE, Fortaleza CE, Brazil

^IMsC

^IIMD, MsC, PhD

^IIIPharmacist, MsC, PhD

^IVMD

^{Address to correspondence}

ABSTRACT

The scurvy shows an inflammatory disease and gingival bleeding. Nevertheless, in an animal model for guinea pigs, described by Den Hartog Jager in 1985, scurvy was associated with a motor neuron disease with demyelinization of the pyramidal tract, provoking neurogenic atrophy of muscles. Aiming at searching the protective role of vitamin C in nervous system, a pharmacological, morphological and behavioral study was conducted. Three experimental groups were used: A100, animals receiving 100 mg/ vitamin C/ day; A5.0, animals receiving 5.0 mg/vitamin C/ day; and A0, animals without vitamin C. We analyzed the weight gain, muscular diameter and behavioral tests. In all tests examined, we found significant differences between the supplemented groups in comparison with scorbutic group (p<0.05). Thereafter, the animals were killed for histopathology of gastrocnemius muscle, spinal cord and tooth tissues. In addition, a morphometric study of periodontal thickness and a-motor neuron cell body diameter were done. The vitamin C-diet free regimen seemed to induce a disruption in spinal cord morphology, involving the lower motor neuron, as confirmed by a significant reduction in neuron perycaria diameter and muscular atrophy, complicated by increased nutritional deficit.

Keywords: vitamin C, ascorbic acid, motor neuron disease, guinea pig, motor behavior.

RESUMO

O escorbuto se caracteriza por doença inflamatória e sangramento gengival. Contudo, num modelo animal em cobaios, descrito por Den Hartog Jager em 1985, o estado escorbútico foi associado à doença do neurônio motor com desmielinização do trato piramidal, provocando atrofia neurogênica dos músculos. Objetivando investigar o papel protetor da vitamina C no sistema nervoso, um estudo farmacológico, morfológico e comportamental foi conduzido. Três grupos experimentais foram usados: A100, animais recebendo 100 mg/vitamina C/ dia; A5,0, animais recebendo 5,0 mg/ vitamina C/ dia; e A0, animais sem vitamina C. Nós avaliamos o ganho de peso, diâmetro muscular e realizamos testes comportamentais. Em todos os testes examinados, detectamos diferenças significantes dos grupos suplementados em relação ao grupo escorbútico (p<0,05). Os animais foram sacrificados para histopatologia do músculo gastrocnemius, medula espinhal e tecidos dentários. Também foi realizado estudo morfométrico da espessura do periodonto e do diâmetro dos corpos celulares dos neurônios motores a. A carência de vitamina C parece comprometer a morfologia da medula espinhal envolvendo o neurônio motor inferior, confirmada pela redução significativa do diâmetro dos pericárions e atrofia muscular, complicada pelo aumento do déficit nutricional.

Palavras-chave: vitamina C, ácido ascórbico, doença do neurônio motor, cobaio, comportamento motor.

Vitamin C is an essential nutrient for human beings and few other species that do not have the enzymatic system L-gulono-g-lactone oxidase, the final step in ascorbic acid biosynthesis pathway, catalyzing the conversion of L-gulono-g-lactone to keto-gulono-g-lactone^1,2. Ascorbic acid is synthesized in the liver of mammals capable of its synthesis or in the kidney of reptiles and amphibians. The ancestors of primates had lost their ascorbate synthesizing ability during the evolution owing to mutations in the gulono-lactone oxidase gene¹. The identification of an animal model for ascorbic acid deficiency was carried out by Holst & Frölich in 1907, who accidentally produced scurvy in guinea pigs and thus provided a useful animal test for studies of the repercussion of vitamin C deficiency.

Ascorbic acid has been implicated in a myriad of biological actions, most of them related to defective collagen synthesis seen in a scurvy status^3,4. Moreover, ascorbic acid participates in a variety of enzymatic reactions (e.g. collagen and catecholamine synthesis) as an electron donor, and it has a role in redox dynamics as one of the most important water-soluble antioxidants of the mammalian tissues^1,5-7. The high concentrations of this compound in central nervous system (CNS), however, suggest that ascorbic acid is a crucial factor for the antioxidant network as a scavenger of reactive oxygen species in intracellular and extracellular compartments, performing a neuroprotective role under physiological and pathological conditions^8-10.

Up to date, the recent new data have showed the role of L-ascorbic acid as an important neuromodulator in glutamatergic transmission by a heteroexchange mechanism in the vicinity of the nervous tissue and so contributing to reduce the glutamate excitotoxic action in synaptic cleft^5,11-13. The altered glutamate metabolism and disruption in carrier proteins, jeopardizing its elimination, can overstimulate excitatory aminoacid receptors, inducing a lethal calcium influx at a post-synaptical level^14,15. Glutamate seems to trigger the release of ascorbate from primary cultures of cerebral astrocytes, which are the most abundant cell in the brain¹⁶. There is a increasing evidence that the specific loss of GLT-1 glutamate transporter in astroglial cells could contribute or even figured as a direct causal factor leading to disruption of motor neurons¹². In addition, the glucose and glutamate transport impairment in cortical synaptosomes from Cu/Zn-SOD mutant mice exacerbated ischaemic brain injury by a possible toxic product, generated from the mutant. That toxic action could be increased by the oxidative insult^10,17.

The scurvy status shows as a cardinal manifestation an inflammatory disease with periodontal apparatus involvement^18,19. Nevertheless, in an experimental animal model described by Den Hartog Jager (1985)²⁰, the scurvy picture was associated with a motor neuron disease, with demyelinization of the pyramidal tract and consequent muscular atrophy. Aiming at investigating the vitamin C deficiency impact in the decline of motor abilities, we carried out a pharmacological, behavioral and morphological study in guinea pigs, also comparing the benefice of ascorbic acid supplementation.

METHOD

Materials and treatment regimen

The subjects were young adult guinea pigs (250-350 g) obtained from HEMOCE (Hematology and Hemotherapy Center of Ceará, Northeastern Brazil). They were housed in individually cage in an animal holding room, installed in Department of Physiology and Pharmacology of Federal University of Ceará, with 12 L : 12 D cycle, food and water were available ad libitum.

During the experimental period, the guinea pigs were fed with an ascorbate deficient diet (Fri-Ribeâ; see Table 1). The animals were randomly divided into three experimental groups. A100 group (n=8), with animals receiving 100 mg/vitamin C/day; A5.0 group (n=8) with animals receiving 5.0 mg/vitamin C/day and A0 (n=8), vitamin C diet-free group. The supplementation was carried out by orogastric probe, immediately after its preparation. The experimental time course was 26 days, (enough time to induce the scurvy state in non supplemented guinea pigs).

Experimental devices and protocols

Motor parameter - During two days intervals, it was done measures of the muscular right thigh diameter by an electronic digital caliper (Starrett^®), just bellow the coxo-femoral joint as a muscular activity paradigm in vivo, with careful proceeding to avoid the muscular mass compression.

Behavioral analysis - In the end of the 26 days, motor behavior tests were conducted in a large open box (50x50x 25 cm) with a transparent Perspex front panel. In order to facilitate the behavioral measurements, animals were videotaped (Panasonic, NV-RJ16BR, video camera with zoom lens) in a dark and silent room. The video camera was positioned directly in front of the Perspex window at the front of the box. Videotapes were played using a Panasonic NV-SD video recorder. Animals from the experimental groups were videotaped automatically for 15 min. In absence of people in the room, three behavioral variables were measured for all animals: (1) Climbing, (2) Running and (3) Rearing. All observations displayed in a video monitor were analyzed in a IBM-PC computer with a MS-DOS program named Comporta, designed for behavioral testing by Prof. Marcus R. Vale from Federal University of Ceará, where a standardized key from computer keyboard yielded a behavior pattern registry.

Autopsy procedures - Guinea pigs were killed under chloral hydrate anesthesia by formaldehyde infusion just after a carotida cannulation. The direct infusion in blood stream aimed to improve the fixation quality of the specimens. Samples were taken from the gastrocnemius muscle, superior incisors with maxillary bone and from the spinal cord, at the thoracic level, after tissue-wash in physiological solution.

Morphology approach (Morphometry)

Motor neurons - We analyzed the spinal cord slices using a light binocular microscope with millimeter ocular (Leitz Wetzlar Germany Periplan, GF 10x). The slices were stained by cresyl violet technique to detect the basophilic material called Nissl substance in a-motor neuron in anterior gray horn. We measured the cell body diameter of 100 spinal motor neurons in a set of five serial cross-sections from each animal (n=5) along the experimental group, searching the most conspicuous ones in great magnification.

Periodontal membrane - We analyzed the periodontal ligament thickness in decalcified tooth stained by Van Gieson and Mallory's trichrome in three different levels: (a) at the level of alveolar ridge; (b) at the root intermediate level; (c) at the most apical radicular level. We examined 100 specimens in a set of five serial cross-sections at thoracic level from each animal (n=5) along the experimental group. The mean of these three level measurements was obtained as described above.

Histopathological technique - Tissue samples were obtained post-mortem, fixed in buffered formalin and embedded in paraffin. Six- micrometer-thick sections from the paraffin-embedded blocks were hydrated after removing the paraffin, stained and mounted by entellan medium in glass slides. In the case of Weil's method, 10 micrometer-thick sections were used. Hematoxylin-Eosin technique was chosen to study muscle histology, Van Gieson and Mallory' trichrome to tooth specimens, and Weil and cresyl violet to spinal cord samples. The Weil's method was useful to detect the integrity of pyramidal motor tract myelinization.

Statistics - Statistical analysis was performed using GraphPad InStat™ for MS-DOS and GraphPad Prism™ for MS-Windows on an IBM-PC. The statistical significance of the differences was analyzed by Student-Turkey and ANOVA for continuous variables. When data violated assumptions for parametric tests, non-parametric Mann Whitney U-test was used. Significance was considered when p<0.05.

RESULTS

Weight gain - The supplemented animals showed weight gain during all the experimental period, in a dose relationship. The scorbutic animals exhibited weight gain just during the first two weeks (until about 12^th day), thereafter they showed a remarkable weight loss, especially in the final days of the protocol (Fig 1).

Clinical state and autopsy findings - Considering the scorbutic rating scale designed for guinea pigs by Verlangieri et al.²¹, clinical signs included hemorrhages, reduction of exploratory behavior in open field, back/neck piloerection, weakness, ataxia, dysbasia and weight changes were in some degree observed particularly in the last days of the experiment. We also detected spontaneous bone fracture and dystrophy frequently in anterior thighs (in a few animals). The autopsy findings confirmed the subcutaneous, subepimysial and joint hemorrhages, further hemorrhagic gastroenteritis, bone and tooth fragility and muscular atrophy were the more obvious clinical features.

Muscular diameter - The supplemented and non-supplemented animals showed a progressive gain of muscular mass, during the experimental period. However, we detected a significant reduction in right thigh muscular diameter, measured by digital caliper, in the vitamin C deficient animals in the last days of the experiment in comparison with the supplemented groups (Fig 2). The A100 and A5.0 groups showed a mean muscular diameter significantly superior to the deficient group (p<0.001 and p<0.05, respectively).

Motor behavior examination - We found in scorbutic animals a relevant decrease in motor behavior in comparison to the control groups in all tests examined, which had been corroborated by the prior clinical findings (Fig 3). We noted almost a completely non-existence of running behavior in A0 animals. A100 and A5.0 supplemented control groups exhibited no significant difference, concerning all motor behaviors studied (p<0.05).

Light microscopy (morphometry)

Periodontal ligament - The non-supplemented group exhibited a decrease of mean thickness of the periodontal ligament (PL) as compared with A100 and A5.0 groups (p<0.001 and p<0.05, respectively). The periodontal ligament reduction was observed in a variable degree through all its extension since alveolar ridge level until its more apical process (Fig 4).

a-motor neurons - We confirmed a significant reduction in the cell body mean diameter of a-motor spinal neurons (p<0.001) when compared with the supplemented groups. There was no statistically difference in measurements between A100 and A5.0 supplemented groups (Fig 5).

Histopathological changes

Skeletal muscle - The gastrocnemius muscle from scorbutic animals (Figs 6-A and ^B) appeared atrophied in small groups as compared to controls, exhibiting muscle fibers with irregular shapes, including oval and angular aspects, among hypertrophied ones. Lack of cylindrical cell morphology and normal arrangement were conspicuous in some fasciculi. Indeed, severe reduction of muscle fiber diameter and incompletely cross-striations were also observed. We confirmed micro hemorrhagic areas in supporting tissues.

Spinal Cord - The slices of spinal cord at thoracic level from A0 animals confirmed the signs of ventral horn motor neuron involvement, as illustrated by chromatolysis features as seen by cresyl violet staining method (Fig 7.1-B and ^D) as compared to controls (Fig 7.2-E). Furthermore, a relevant number of motor neurons showed cell body diameter decrease and hyaline inclusions (Fig 7.1-B). Some a-motor neurons almost totally lack their morphological polarity. Some spinal cord specimens exhibited a reactive gliosis, with marked conspicuous satellitosis. However, the guinea pig pyramidal tract was preserved in A0 subjects as seen by Weil's myelin staining at the level of posterior funiculus of white matter (Fig 7.1-A). The reduction of Weil staining contrast in the gray matter of anterior columns from scorbutic spinal cords revealed decreased cytoplasmatic extensions from motor neurons (^{Fig 7.1-C}) as compared to controls (Fig 7.2-F), unless without complete disappearance. There was no important alteration in comissural white matter neither others fasciculi areas (Fig 7.2-G).

DISCUSSION

The recent findings about the compartmentalization of brain ascorbate and glutathione (GSH) between neurons and glia indicated that these compounds, because of their antioxidant properties, have great relevance in homeostatic control of oxidative products in CNS^12,22-24. These data suggest that ascorbate is predominantly localized in neurons. Their tissue contents are region-specific and in the milimolar range. This scenery renders the crucial role of ascorbate in several physiological and pathological conditions at the vicinity of the CNS. Indeed, it reflects the oxidative metabolism in neurons at mitochondria level. Motor neuron vulnerability to oxidative stress is credited to low intracellular calcium sequestration and high energy dynamics, thus imbalance of free radical buffering by increased production of oxygen species and defective antioxidant capacity, taking together, could undergo toward a growing impairment²⁵.

Ascorbate is released from intracellular sites in close association with the glutamate uptake by a heteroexchange mechanism within glutamatergic neurons and glia. This ascorbate delivery in CNS motor tracts under synaptic firing might be neuroprotective. The overall glutamate metabolism seemed to be affected by motor neuron disease, triggering a degenerative process by an amplified loop involving a large number of well documented lethal conditions induced by several enzymatic actions, including endonucleases, NOS and peroxidase pathways, finally leading to membrane lipid peroxidation and cell death^17,26.

The oxidative motor neuron injury explained the importance of antioxidant network balance both at water and lipid cell phase. Ascorbic acid may be a useful tool in recycling a-tochoferol and glutatione radical back to the reduced form as an electron donor in CNS, playing an indirect protective action²⁷. Moreover, recent studies have demonstrated that the ascorbic acid is beneficial against peroxynitrite (ONOO-) radical attack, which is closely related to motor neuron damage^28,29. In addition, GLT-1 glutamate protein carrier system may become jeopardized by oxygen reactive species, thus causing its progressive accumulation on synaptic cleft, over stimulating excitatory aminoacid receptors on post-synaptic dominium.

The guinea pig nutritional model for experimental motor neuron disease, first described by Den Hartog Jager²⁰, was strongly argued by Sillevis Smitt & De Jong³⁰, who used a chronic ascorbic acid deficiency protocol and a purified diet containing minimal and defined amounts of AA, based on the recommendation of the National Council Research. Their results did not show changes in spinal pyramidal tract, although, they confirmed a nutritional myopathy, complicated by trauma, and arthrogenic factors without apparently signs of anterior horn motor neuron death³¹. These authors suggested that Den Hartog Jager's previous findings were defined from an inappropriate dietary regimen to experimental guinea pigs. Shils³² stated the importance of adequate control groups in nutrition studies.

In light of an actual knowledge, the reduction of the net scavenger milieu on spinal cord from scorbutic guinea pigs could be quite intensified by the assumption that, under poor neuron density environments such as those found in more posterior areas of the CNS, there would be diminished vitamin C storage capacity (white matter: gray matter tissue ratio), for extracellular scavenge requirements. In addition, the high metabolic motor nervous cells seemed to be more prone to oxidative insults. Recently, it was confirmed in a G93A transgenic mice model that mutation of SOD1 elevates the level of both H₂O₂ and OH but not in the mice, overexpressing normal human SOD1³³. Moreover, the combined treatment with trientine, a chelating agent for cooper, and ascorbate had been beneficial in familial amyotrophic lateral sclerosis model mice, with a protective effect on the onset of the disease and its end-point³⁴.

Whether the vitamin C withdrawal had a direct causal action in the motor neuron impairment or whether only had a secondary role in an multifactorial process, we did not know. Our results showed that on scurvy status, the guinea pigs exhibited a motor impairment probably induced by free radical overload and this rich oxidative scenario seemed to trigger the progressive motor neuron jeopardizing, even though without significant motor neuron death, but size reduction and chromatolysis, showing the vulnerability of a-motor neuron under vitamin C environmental collapse in the vicinity of nervous tissue. Muscle weakness was linked to clinical carnitine deficiency seen in scurvy³⁵.

In our assessment, we choose an intermediate diet, containing vitamin A 10,000 IU/Kg, zinc 0,1 mg/kg, copper 10 mg/kg and manganese 50 mg/kg. In this chronic deficiency protocol, in turn, we detected an progressive disruption in motor neuron unit, under drastic affected animals, even though, without pyramidal tract involvement. The decline of motor neuron viability seemed to be restricted to spinal cord level, as confirmed by morphometric analysis and histopathological picture, since we did not find important alterations in other CNS regions like cerebral and cerebelar cortices (data not shown). Since there was no apparently sign of pyramidal tract degeneration, which in guinea pigs is localized at the base of posterior column in spinal cord^20,31, neither upper motor neuron involvement in our protocol, the present results do not support the previous idea of an animal model for lateral amyotrophic sclerosis as described by Den Hartog findings. The decline of all motor skills investigate seemed to be directed related to increasing ascorbic acid default, complicated by a myriad of scurvy outcomes, most of them involving skeletal tissues, however, the vitamin C megadosis seemed not to significantly ameliorate the motor abilities tested because there was no important difference in the motor behaviors assisted between both supplemented groups.

Acknowledgment - The authors would like to thank José Ivan Rodrigues de Sousa and Oséas Ribeiro Queiroz for technical assistance and Sílvio Alves Costa for his aid in the electronic typewriting of this manuscript.

Received 19 June 2002, received in final form 12 September 2002.

Accepted 17 September 2002.

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Publication Dates

• Publication in this collection
  16 Apr 2003
• Date of issue
  Mar 2003

History

• Received
  19 June 2002
• Accepted
  17 Sept 2002