Vascular insult accompanied by overexpressed heme oxygenase-1 as a pathophysiological mechanism in experimental neurolathyrism with hind-leg paraparesis.

Neurolathyrism (NL) is a motor neuron disease characterized by spastic paraparesis in the hind legs. ß-N-oxalyl-l-α,ß-diaminopropionic acid (l-ß-ODAP), a component amino acid of the grass pea (Lathyrus sativus L.), has been proposed as the cause of this disease. In our NL rat model, we previously reported that transient intra-parenchymal hemorrhage occurred in the lower spinal cord during the early treatment period. We show here a possible pathological role of the hemorrhage in motor neuron damage and paraparesis pathology. In the lumbo-sacral spinal cord, blood vessel integrity was lost with numerous TdT-mediated dUTP nick end-labeling-positive blood vessel-like structures occurring simultaneously with the hemorrhage. We observed a coincident >10-fold increase in heme oxygenase-1 (HO-1) only in the lower spinal cord. The early period of paraparesis in the lower leg was greatly suppressed by pretreatment with zinc protoporphyrin IX, a HO-1 inhibitor. In vitro, l-ß-ODAP was toxic to human umbilical vein endothelial cells compared to l-glutamate. The present data shed light on the role and the mechanism of vascular insult in causing dysfunction and moribund motor neurons in experimental NL.

Lathyrism and Socioeconomic Disparities: A Neglected Public Health Problem in Northeast Ethiopia.

Lathyrism is an incurable neurological disorder, resulting from excessive consumption of grass pea (Lathyrus sativus), which clinically manifests as paralysis of lower limbs. Because of the high production of grass peas, a large number of people are expected to be affected by the disease in Northeast Ethiopia. However, there is no comprehensive study that quantified the magnitude of the problem. Therefore, in this study, we determined the prevalence of lathyrism and socioeconomic disparities in Northeast Ethiopia. A community-based cross-sectional study was used which used a quantitative method of data collection from January to February 2019. Data were collected from a total of 2,307 inhabitants in the study area using structured questionnaires. Lathyrism cases were identified using a case definition of symmetrical spastic leg weakness, and subacute or insidious onset, with no sensory deficit, and with a history of grass pea consumption before and at the onset of paralysis. The majority (56.8%) of participants were male, and 34.7% were aged 45 years or older. Overall, the prevalence of lathyrism was 5.5%, and it was higher in males (7.9%) than in females (2.5%). Moreover, the prevalence was higher among farmers (7.0%) than merchants (0.3%), very poor economic status (7.2%) than very rich (1.1%), who produced (9.6%) grass pea than not produced (0.9%), and those who used clay pottery (6.2%) than metal (4.8%) for cooking. The prevalence of lathyrism in Northeast Ethiopia is remarkably high. Therefore, we recommend lathyrism to be among the list of reportable health problems and incorporated in the national routine surveillance system.

[Research in Motor Neuron Diseases Caused by Natural Substances: Focus on Pathological Mechanisms of Neurolathyrism].

Diseases of the motor-conducting system that cause moving disability affect socio-economic activity as well as human dignity. Neurolathyrism, konzo, and amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC) have attracted researchers to study the pathology of motor neuron (MN) diseases such as ALS. I have been studying neurolathyrism, which is caused by overconsumption of a legume grass pea (Lathyrys sativus L.). Among people who consume the legume as a food staple, many developed life-long paraparesis in their legs. ß-N-oxalyl-l-α,ß- diaminopropionic (l-ß-ODAP; BOAA), contained in this plant, is a neurotoxic analog of l-glutamic acid. We have clarified that in addition to the causal involvement of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamatergic receptor in MN death, a toxic role of group I metabotropic glutamate receptors as well as transient receptor potential channels were involved in the MN insult by l-ß-ODAP using primary MN culture. We have also established a neurolathyrism rat model by repeated, peripheral l-ß-ODAP treatment to newborn rats under mild stress. Rats showing hind-leg paraparesis with an incidence rate of around 25% were useful to study the in vivo pathology of MN disease. MNs of these rats were greatly decreased at their lumbo/sacral segments at various ages. Intra-parenchymal hemorrhage was consistently observed in paraparetic rats but not in cripple-free, treated rats. MN were depleted even at an acute period around bleeding spots, suggesting catastrophic neuro-vascular-glial interaction in this MN disease. Summaries of konzo and ALS-PDCs studies are also introduced.

Stress-related over-enhancement of the hypothalamic-pituitary-adrenal axis causes experimental neurolathyrism in rats.

Neurolathyrism is a motor neuron disease that is caused by the overconsumption of grass peas (Lathyrus sativus L.) under stressful conditions. The neuro-excitatory ß-N-oxalyl-L-α,ß-diaminopropionic acid present in grass peas was proposed the causative agent of spastic paraparesis of the legs. Historical reports of neurolathyrism epidemics, studies of neurolathyrism animal models, and in vitro studies on the mechanism of ß-N-oxalyl-L-α,ß-diaminopropionic acid toxicity support the hypothesis that stress increases susceptibility to neurolathyrism. To elucidate the role of stress in neurolathyrism-induced motor dysfunction, we focused on the hypothalamic-pituitary-adrenal axis in a rodent model of neurolathyrism. Our results implicated increased glucocorticoid and neuroinflammation in the motor dysfunction (paraparesis) exhibited by the stress loaded rat models of neurolathyrism.

Reduced reciprocal inhibition is seen only in spastic limbs in patients with neurolathyrism.

If reduced reciprocal inhibition plays a causal role in the pathophysiology of spasticity as has been suggested in several studies, the inhibition is expected to be impaired in spastic, but not in normal muscles. Patients with neurolathyrism offer a possibility of testing this prediction since the spastic symptoms in these patients are restricted to the lower extremities only. Three patients with neurolathyrism were tested. Their data were compared with 15 age-matched healthy subjects. All patients showed signs of spasticity in the legs. Two patients had normal voluntary muscle force in the lower extremities and one had decreased force. No clinical abnormalities were found in the upper extremities. Reciprocal inhibition between ankle dorsiflexor and plantarflexor muscles was absent in all patients, whereas the inhibition between wrist extensor and flexor muscles was present and of normal size and latency. These findings are consistent with the hypothesis that reduced reciprocal inhibition plays a causal role in the pathophysiology of spasticity.

Periodontal & systemic bone changes in rats with experimental lathyrism.

BACKGROUND & OBJECTIVE: It is not clear how lathyrism affects the systemic bone metabolism. We therefore undertook a study to observe periodontal and systemic bone changes by performing radiological, metabolic, and bone densitometric evaluations in rats with experimental lathyrism. METHODS: A total of 30 rats were used. Experimental lathyrism was induced by once daily subcutaneous administration of beta-aminopropionitrile (beta-APN), at a dose of 5 mg beta-APN/0.4 ml per 100 g of body weight for 40 days. After 40 days, vertebral bone mineral density was analyzed by means of dual energy X-ray absorbtiometry in both groups. Blood was drawn by cardiac puncture and the animals were decapitated. Serum calcium levels were measured. Right mandibles were removed and radiographs were obtained. Alveolar bone level was determined in the radiographs. RESULTS: In all lathyritic rats, alveolar bone level was pathologically decreased with visible resorption. Vertebral bone mineral density values of lathyritic rats did not differ significantly from those of the control group. Compared to controls, there was a statistically significant decrease in serum calcium levels in the lathyritic group (P<0.001). INTERPRETATION & CONCLUSION: Significant alveolar bone resorption without alterations in vertebral bone mineral density indicated that lathyrogen administration for 40 days presumably has not caused systemic demineralization. This model could be used for studying the role of local and systemic agents on periodontal alveolar bone resorption.

Determination of systemically & locally induced periodontal defects in rats.

BACKGROUND & OBJECTIVE: The role of lathyrogens on bone metabolism is unclear, therefore we undertook this study to observe periodontal and systemic alterations in experimental lathyrism in rat and compare these changes to that observed in the locally induced periodontitis group. METHODS: A total of 45 male Wistar rats were equally divided in the lathyritic group (group 1), ligature-induced periodontitis group (group 2), and healthy controls (group 3). Experimental lathyrism was induced by once daily subcutaneous administration of beta-aminoproprionitrile (beta-APN), at a dose of 5 mg/0.4 ml per 100 g of body weight for 40 days. Ligature-induced periodontitis was created by tying silk ligatures on the necks of mandibular molars. After 40 days, blood samples were obtained and the animals were decapitated. Radiographic observations, extraction tests, histologic evaluations were performed, and serum ALP activity and gingival tissue IL-1beta levels were measured. RESULTS: Significant alveolar bone resorption around the mandibular molar teeth (P<0.001); lower extraction force levels (P<0.001); higher numbers of lymphocytes and macrophages (P<0.01) (both in connective tissue and epithelium at the dentogingival junction); decreased ALP activity (P<0.001); and increased gingival tissue IL-1beta levels (P<0.001) were observed in groups 1 and 2, compared to those in group 3. ALP activity was higher in group 1 than in group 2 rats (P<0.05). INTERPRETATION & CONCLUSION: Similar radiographical and histopathological findings and comparable increases in gingival tissue IL-1beta levels both in groups 1 and 2 showed that in addition to resorption of alveolar bone, chronic inflammation of periodontium also occurred both in the lathyritic rats as well as in ligature-induced periodontitis group rats.

The central nervous system in a case of neurolathyrism.

We examined the central nervous system of a 67-year-old man who showed symptoms of lathyrism after being imprisoned at age 35 and fed on a diet of chick peas, Lathyrus sativus. The most obvious changes were loss of axons and myelin in the pyramidal tract in the lumbar spinal cord. These alterations correlated with spastic paraparesis. Other changes at the same level were a mild degree of degeneration of anterior horn cells. Pallor of Goll's tracts and axonal swelling in Goll's nuclei were also observed. A few examples of senile plaques and neurofibrillary tangles were observed in Ammon's horn.

Organization of collagen types I and V in the embryonic chicken cornea.

The distribution and organization of type I and type V collagens were studied in the embryonic chicken cornea using anti-collagen, type specific, monoclonal antibodies and immunoelectron microscopy. These studies were performed on lathyritic 17-day corneas treated at 4 degrees C or 37 degrees C. At the lower temperature, collagen fibril structure is disrupted; at the higher temperature, normal fibril structure is maintained. Corneas from non-lathyritic 17-day chick embryos, reacted at the two different temperatures, were studied for comparison. In Bowman's membrane, the thin (20 nm) fibrils were labelled by antibodies against both type I and type V collagen under all conditions studied. In the corneal stroma, the striated collagen fibrils (25 nm) were labelled with the antibodies against type I collagen in all cases, and by antibodies against type V collagen under conditions where fibril structure was disrupted. These results are consistent with the concept of heteropolymeric fibrils consisting of both type I and type V collagen molecules assembled such that the epitopes on the type V molecule are unavailable to antibody unless the fibrillar structure is disrupted. We suggest that the interaction of type V collagen with type I collagen may be responsible for the small diameter fibrils and the rigid control of fibril structure found in the cornea.

Experimental intracranial aneurysms in rats. A gross and microscopic study.

Experimental cerebral aneurysms were induced in rats in which the left carotid artery was ligated. The rats were rendered hypertensive by the Goldblatt procedure, and fed a diet containing beta-aminopropionitrile. These intracranial aneurysms have significant gross and microscopic similarities to human berry aneurysms. The procedures and microscopic observations indicate that this experimental model could be useful for studying the development, pathophysiology, and therapy of saccular aneurysms.

The variation of sonic plesio-velocity in dose dependent lathyritic rabbit femurs.

It has been hypothesized that the origin of the increased elastic modulus of mineralized bone compared with the demineralized bone matrix is in the higher crosslinking density of the collagen in bone. Osteolathyrism is ascribed to an inhibition of crosslinking of the collagen by the lathyrogen and should be accompanied by a decrease in the elastic modulus of the bone. Dose dependent osteolathyrism was induced by varying the amount of BAPN ingested per day by young New Zealand white rabbits until they were mature. The femurs exhibited dose dependent properties, including wet bone density and sonic plesio-velocity in the radial direction. It was found that there is no minimum critical dose. Even though no overt osteolathyritic stigmata could be observed, both the sonic properties and the wet bone density could be affected at any dose level. The sonic plesio-velocity and the longitudinal elastic modulus decreased with BAPN dose level, most rapidly at the lower dose levels and then less so at higher dosages. Since BAPN acts to inhibit the crosslinking density, it appears that the elastic moduli of bone are dependent on the crosslinking density.

Histological study, including ultrastructural quantification, of the periodontal ligament in the lathyritic rat mandibular dentition.

A 0.15 per cent solution of aminoacetonitrile was added to the drinking water of young adult male Wistar rats for 18 days. Their right mandibular incisors were maintained unimpeded by frequent trimming. After 18 days, the periodontal ligaments of the right mandibular incisors and first molars were prepared for light and electron microscopy. For control purposes, similar material was obtained from animals fed on a normal diet ad libitum or pair-fed. With light microscopy, lathyritic incisor periodontal ligaments showed areas of normal connective tissue appearance interspersed with areas of degeneration. In the lathyritic molars, cell-free areas were observed. With electron microscopy, quantification of features in the connective tissue regions of the lathyritic ligament showed little change. In cell-free areas, many collagen fibrils had smaller fibril diameters than normal.

Analysis of stress-strain curves at fast and slow velocities of loading in vitro in the transverse section of the rat incisor periodontal ligament following the administration of beta-aminopropionitrile.

The in vitro mechanical properties of this ligament were examined by analysing the stress-strain curve obtained from a transverse section of the mandible. Mechanical measures were compared between normal rats and lathyritic rats given drinking water containing 0.2% of beta-aminopropionitrile (BAPN) for 20 days, and between the velocity of loading at 10(4) and 1 mm/24 h. The daily dose of BAPN decreased gradually because the body weight increased gradually. At the velocity of 10(4) mm/24 h, the maximum shear stress, elastic stiffness and failure strain energy density in the experimental subgroup fell to 43-50% of the control values, and at 1 mm/24 h to 71-80%. The maximum strains were not significantly different between the control and experimental subgroups either at 10(4) or at 1 mm/24 h. In the control subgroups, the maximum shear stress, elastic stiffness and failure strain energy density at 1 mm/24 h fell to 0.04-0.30% of those at 10(4) mm/24 h, and in the experimental subgroups to 0.08-0.43%. The maximum strains at 1 mm/24 h were 1.7-1.8 times greater than those at 10(4) mm/24 h in both the control and experimental subgroups. It is assumed that changes in the mechanical properties of the periodontal ligament were caused by inhibition of maturation of the periodontal collagen fibres. Assuming that the periodontal ligament is viscoelastic in nature, it is suggested that the main component reacting at 10(4) mm/24 h was an elastic one and that both components, with emphasis on the viscous one, interact at 1 mm/24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Fine structure of the myotendinous junction of lathyritic rat muscle with special reference to connectin, a muscle elastic protein.

The fine structure of the myotendinous junction of the skeletal muscle of lathyritic rats caused by beta-aminopropionitrile was investigated. In the junction there are finger-like processes of muscle fibers, in which thin filaments were extended from the last Z lines of myofibrils and attached to the sarcolemma of the processes. By the heavy meromyosin decoration technique, these thin filaments were identified as actin filaments. In the lathyritic muscle, the thin filaments were markedly fewer in number and distributed sparsely in the sarcoplasm. The content of connectin, an elastic protein, which is localized in myofibrils and also in sarcolemma was significantly decreased in the lathyritic muscle. A possible relationship between the changes in the fine structure of the myotendinous junction and in the connectin contents is discussed.

[Histochemical model investigations of the action mechanisms of lathyrogenic substances]. / Histochemische Modellversuche zum Wirkungsmechanismus lathyrogener Substanzen.

In histochemical model studies, aminoacetonitrile and 3-aminopropionitrile can form hydrolysis-stable azomethines (Schiff bases) with periodate-induced aldehyde groups of tissue slices. Both substances and triethylentetramine do not inhibit the histochemical monoaminoxidase activity, they even can act as substrates of this enzyme. These substances cause an inhibition of the histochemical aminopeptidase M activity, but this inhibition was recognized as a methodological error due to the formation of complexes between diazonium salts and aliphatic amines. The results indicate that the inhibition of the lysyl oxidase will not be the only mechanism of action of lathyrogenic substances.

[Quantitative morphological analysis of bone remodelling in an experimentally-induced skeletal disease (author's transl)]. / Quantitativ-morphologische Untersuchungen des Knochenumbaues am Beispiel einer experimentellen Skeletterkrankung

In the lumbar vertebrae of lathyritic rats bone remodelling was investigated using histomorphometrical and tetracycline labelling methods and a newly developed, computer-aided photometrical quantitation of mineral densities in microradiographs. The morphometrical analysis showed a high-graded decrease in cancellous bone due to increased resorption and cessation of endochondral bone formation. On the other hand, increased formation of new bone in sites of muscular insertion led to the formation of exostoses. Disturbed mineralisation of the newly-formed bone is indicated by increased osteoid volume and diffuse labelling with tetracycline. These findings were confirmed by the quantitation of the mineral densities. The mineralisation defect is related to the disturbed collagen synthesis in lathyrism.

Comparison of the effects of two lathyrogens on the reproductive system of the laying hen.

The effects of two lathyrogens (beta-aminopropionitrile and semicarbazide) on the reproductive system of the laying hen were compared by measuring the changes in the ovary and oviduct associated with a diminished egg production rate. The laying hens were fed diets containing either beta-aminopropionitrile, the toxic constituent of certain legume seeds in the genus Lathyrus or semicarbazide to induce lathyrogenic effects in which reduced egg production was one of the toxic effects. The gross examination of the reproductive system of the hens revealed that ovarian activity was greatly impaired. The reduced egg production after feeding beta-aminopropionitrile was due to the reduced growth rate of the ovary, and in the case of semicarbazide, to the atresia of large yolky follicles.

Experimental lathyrism: exostoses and aneurysmal-like bone cysts of the mandible in the rat.

The effect of chronic lathyrism on the mandible of the rat was studied. Exostoses, thinning and discoloration of the cortical plates, intraosseous bleeding and hemorrhagic cyst were observed after six weeks of administration of beta-aminoproprionitrile fumarate. The exostoses consisted of homogeneous cellular fibrous tissue containing osteoid, hyalin-like material and abundant ground substance. The cysts were filled with erythrocytes and lined by spindle-shaped fibroblasts, collagen fibers or osteoblast-like cells. Between the cysts, bands and sheets of dense and cellular connective tissue presenting foci of hemorrhage, osteoid and metaplastic cartilage were found. Numerous mast cells were demonstrated with alcian and toluidine blue in the hematopoietic bone marrow, cancellous bone and around the capillary network surrounding the exostoses and cysts. Exostoses have already been studied and explained by the action of BAPN (lysyl oxidase). Hemorrhagic cysts could be explained by histamine release from the mast cells (hypervascularity, hyperemia, increased permeability, rupture and progressive confluence of these small hemorrhagic cysts). Histopathology of the experimental cysts had some points in common with the human aneurysmal bone cyst. Could the pathogenic mechanisms be similar for both lesions?

New insights into the mechanism of neurolathyrism: L-ß-ODAP triggers [Ca2+]i accumulation and cell death in primary motor neurons through transient receptor potential channels and metabotropic glutamate receptors.

Neurolathyrism is a motor neuron (MN) disease caused by ß-N-oxalyl-L-α,ß-diaminopropionic acid (L-ß-ODAP), an AMPA receptor agonist. L-ß-ODAP caused a prolonged rise of intracellular Ca(2+) ([Ca(2+)]i) in rat spinal cord MNs, and the [Ca(2+)]i accumulation was inversely proportional to the MN's life span. The [Ca(2+)]i rise induced by L-ß-ODAP or (S)-AMPA was antagonized completely by NBQX, an AMPA-receptor blocker. However, blocking the L-type Ca(2+) channel with nifedipine significantly lowered [Ca(2+)]i induced by (S)-AMPA, but not that by L-ß-ODAP. Tetrodotoxin completely extinguished the [Ca(2+)]i rise induced by (S)-AMPA or kainic acid, whereas that induced by L-ß-ODAP was only attenuated by 65.6±6% indicating the prominent involvement of voltage-independent Ca(2+) entry. The tetrodotoxin-resistant [Ca(2+)]i induced by L-ß-ODAP was blocked by 2-APB, Gd(3+), La(3+), 1-(ß-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF-96365) and flufenamic acid, which all are blockers of the transient receptor potential (TRP) channels. Blockers of group I metabotropic glutamate receptors (mGluR I), 7-(hydroxyiminocyclopropan[b]chromen-1α-carboxylate ethyl ester (CPCCPEt) and 2-methyl-6-(phenylethynyl)-pyridine (MPEP) also lowered the [Ca(2+)]i rise by L-ß-ODAP. MN cell death induced by L-ß-ODAP was prolonged significantly with SKF-96365 as well as NBQX. The results show the involvement of TRPs and mGluR I in L-ß-ODAP-induced MN toxicity through prolonged [Ca(2+)]i mobilization, a unique characteristic of this neurotoxin.

Role of glutamate and nitric oxide in onset of motor neuron degeneration in neurolathyrism.

Neurolathyrism is associated with a complex pattern of alterations in the glutamatergic system of the cortical motor region of brain. It is a neurological disorder consorted with excessive consumption of Lathyrus sativus (Grass pea), comprising large amounts of the neurotoxin, ß-N-oxalyl-L-α,ß-diaminopropionic acid (ODAP). ODAP being a potent agonist of ionotropic glutamate receptors enhances their activity and also blocks the astrocytic glutamate/cystine transporters, abutting the neurons. This leads to the sustained increase in the concentration of Glutamate in the synapse which triggers excitotoxicity. L. sativus also contains high levels of arginine and homoarginine which are natural substrates of nitric oxide production, when NO levels increases, it forms peroxynitrite radicals which cause irreparable damage to mitochondria and cellular macromolecules leading to motor neuron degeneration. This review brings together all the molecular events reported so far, emphasizing on the possible role of glutamate and nitric oxide mediated cell death.