Galactose Intoxication Research Articles

N-Glycan Abnormalities in Children with Galactosemia

Galactose intoxication and over-restriction in galactosemia may affect glycosylation pathways and cause multisystem effects. In this study, we describe an applied hydrophilic interaction chromatography ultra-performance liquid chromatography high-throughput method to analyze whole serum and extracted IgG N-glycans with measurement of agalactosylated (G0), monogalactosylated (G1), and digalactosylated (G2) structures as a quantitative measure of galactose incorporation. This was applied to nine children with severe galactosemia (genotype Q188R/Q188R) and one child with a milder variant (genotype S135L/S135L). The profiles were also compared with those obtained from three age-matched children with PMM2-CDG (congenital disorder of glycosylation type Ia) and nine pediatric control samples. We have observed that severe N-glycan assembly defects correct in the neonate following dietary restriction of galactose. However, treated adult galactosemia patients continue to exhibit ongoing N-glycan processing defects. We have now applied informative galactose incorporation ratios as a method of studying the presence of N-glycan processing defects in children with galactosemia. We identified N-glycan processing defects present in galactosemia children from an early age. For G0/G1, G0/G2, and (G0/G1)/G2 ratios, the difference noted between galactosemia patients and controls was found to be statistically significant (p = 0.002, 0.01, and 0.006, respectively).

Ultrastructural Changes in the Myelinated Nerve Fibers of the Sciatic Nerve in Galactose Intoxication in Rats

Ultrastructural Changes in the Myelinated Nerve Fibers of the Sciatic Nerve in Galactose Intoxication in Rats

Involvement of endoplasmic reticulum stress in a novel Classic Galactosemia model

Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) activity in humans leads to a potentially lethal disorder called Classic Galactosemia. It is well known that patients often accumulate high levels of galactose metabolites such as galactose-1-phosphate (gal-1-p) in their tissues. However, specific targets of gal-1-p and other accumulated metabolites remain uncertain. In this study, we developed a new model system to study this toxicity using primary fibroblasts derived from galactosemic patients. GALT activity was reconstituted in these primary cells through lentivirus-mediated gene transfer. Gene expression profiling showed that GALT-deficient cells, but not normal cells, responded to galactose challenge by activating a set of genes characteristic of endoplasmic reticulum (ER) stress. Western blot analysis showed that the master regulator of ER stress, BiP, was up-regulated at least threefold in these cells upon galactose challenge. We also found that treatment of these cells with galactose, but not glucose or hexose-free media reduced Ca 2+ mobilization in response to activation of Gq-coupled receptors. To explore whether the muted Ca 2+ mobilization is related to reduced inositol turnover, we discovered that gal-1-p competitively inhibited human inositol monophosphatase (hIMPase1). We hypothesize that galactose intoxication under GALT-deficiency resulted from accumulation of toxic galactose metabolite products, which led to the accumulation of unfolded proteins, altered calcium homeostasis, and subsequently ER stress.

Decreased accumulation of endogenous brain-derived neurotrophic factor against constricting sciatic nerve ligatures in streptozotocin-diabetic and galactose-fed rats

Anterograde and retrograde trafficking of brain-derived neurotrophic factor (BDNF) was examined in streptozotocin-diabetic and galactose-fed rats by measuring accumulation of endogenous neurotrophin proximal and distal to two constricting sciatic nerve ligatures and by direct injection of radiolabeled neurotrophin into the sciatic nerve. Compared to controls, accumulation of endogenous BDNF proximal and distal to the ligatures as well as basal levels in non-ligated nerve segments were decreased in streptozotocin-diabetic and galactose-fed rats. Neither streptozotocin diabetes nor galactose intoxication affected the amount of 125I-labeled BDNF retrogradely transported to the DRG after injection into the sciatic nerve. These results suggest that reduced anterograde and retrograde accumulations of BDNF in experimental diabetes are not a result of impaired capacity for receptor-mediated transport.

Nerve conduction velocity, laser Doppler flow, and axonal caliber in galactose and streptozotocin diabetes

In an initial study, the effects of galactose intoxication on nerve laser Doppler blood flow (NLDF) and nerve conduction velocity (NCV) were assessed after 1–16 weeks of galactose feeding in pentobarbital-anesthetized rats. NLDF was not significantly changed at any time point. NCV was significantly reduced after 16, but not 1 or 4, weeks of galactose feeding. In a second study, NLDF was not significantly changed by 4 weeks of galactose intoxication, but streptozotocin-diabetic NLDF was significantly reduced compared to both control ( P<0.001) and galactose-intoxicated rats ( P<0.05). Compared to control animals, sciatic motor NCV was significantly ( P<0.001) reduced in the galactose group, while sciatic and saphenous sensory NCVs were not significantly changed. In the streptozotocin-diabetic rats, motor and sensory NCVs were all significantly reduced ( P<0.001). In contrast to the NCV findings, mean caliber of myelinated axons in both the saphenous and sciatic nerves was reduced in galactose-intoxicated, but not streptozotocin-diabetic rats. The observed sequence of changes associated with these two models of diabetic neuropathy is not consistent with the proposed roles of ischemia and axonal dwindling in the reported nerve conduction deficits.

BDNF attenuates functional and structural disorders in nerves of galactose-fed rats.

Galactose intoxication of rats was used to disrupt metabolism of Schwann cells and skeletal muscle, two sites that contain the polyol-forming enzyme aldose reductase (AR). Galactose-fed rats develop a neuropathy characterized by nerve conduction deficits and axonal atrophy. To investigate the possibility that galactose metabolism by AR influences axonal function and structure by altering production of neurotrophic factors, the impact of galactose intoxication on nerve and muscle BDNF levels and the effects of exogenous BDNF treatment on galactose neuropathy were examined using biochemical, electrophysiologic and morphometric techniques. Galactose feeding increased BDNF protein in peripheral nerve and muscle. Exogenous BDNF treatment attenuated motor nerve conduction velocity deficits in the sciatic nerve of galactose-fed animals and myelin splitting of motor axons in the ventral root. In contrast, sensory nerve conduction velocity (SNCV) deficits in the sciatic nerve and myelin splitting in the central projections of sensory neurons were not prevented by BDNF treatment. BDNF treatment did not attenuate reduced axonal caliber in the sciatic nerve, but did ameliorate the diminution of the caliber of central sensory projections in the dorsal root. These findings point to the potential use of BDNF in the treatment of peripheral neuropathies.

Schwann cell changes induced as early as one week after galactose intoxication

In galactose neuropathy, aldose reductase inhibitor (ARI)-preventable Schwann cell injury has been reported in studies in which galactose feeding continued over a period of months. Given the link between these morphologic changes and polyol pathway flux, polyol accumulation after just days of galactose feeding points to the possibility that structural changes occur much earlier than previously reported. The aim of this study was to examine rat sciatic nerve after 7 days of galactose feeding for evidence of myelinated fiber injury and establish whether it is related to polyol accumulation. Compared to control or ARI-treated galactose-fed rats, nerves from untreated galactose-fed rats had increased water (P < 0.05) ad dulcitol (P < 0.008) content and decreased amounts of myo-inositol (P < 0.01). Electron microscopy revealed reactive Schwann cell changes in myelinated fibers characterized by increased cytoplasmic volume, and the occurrence of lipid droplets pi granules of Reich and enlarged mitochondria. Dystrophic accumulation of intermediate filaments was also observed in the inner glial loop. Degenerative changes included periaxonal swelling, enlarged mitochondria without recognizable cristae, lysis of Schwann cell cytoplasm and demyelination. Reactive (P < 0.05) and degenerative (P < 0.01) changes as well as the number of redundant basal lamina profiles (P < 0.05) were significantly more frequent in untreated galactose-fed rats compared to controls. ARI treatment attenuated these changes. Consistent with the initial stages of onion-bulb formation, profiles with imbricate Schwann cells were also seen only in untreated galactose-fed rats. The findings suggest that short-term increases in polyol pathway activity can have deleterious effects Schwann cells of myelinated fibers.

Mast Cell Interactions with the Nervous System

In summary, mast cell interactions in the nervous system are relevant to both physiological processes (i.e. reproduction) and pathologic states (i.e. inflammatory demyelination, painful disorders, toxic and metabolic disease, and tumor angiogenesis). Their physiologic roles may contribute to gender-related vulnerability to inflammatory disease and may modulate sensitivity to pain. Mast cells are universally involved in tissue repair and they release and respond to trophic factors such as NGF. These cells also produce and react to cytokines, and thus appear to play a role in tissue degeneration as well as repair. In certain neurological diseases, i.e. multiple sclerosis and Guillain-Barré syndrome, the ability of mast cell proteases to degrade specific myelin proteins suggests that these cells are agents, rather than bystanders, in the demyelinative process. Even more intriguing is their recently identified capacity to process bacterial antigen as efficiently as activated macrophages, suggesting that a more critical role than previously suspected might be considered for mast cells in CNS and PNS demyelination. In experimental metabolic disorders such as galactose intoxication and thiamine deficiency, mast cells appear to play a pathogenic role. Thus, in galactose intoxication, altered BNB vascular permeability occurs in conjunction with mast cell proliferation and degranulation, while in thiamine deficiency, increased histamine levels have been reported in the rat thalamus (79) and are associated with cell death and proliferation as well as mast cell degranulation (Powell and Langlais, unpublished observations). Structural interactions between mast cells and a variety of other cells have been observed, as well as close approximation of mast cells to nerve endings in tissues in which mast cells are especially active. Due to their paracrine nature, mast cells can modulate events in their microenvironment through explosive degranulation, piecemeal degranulation, or "transgranulation" as they insert granules into neighboring cells. Lastly, these cells play specific roles in reparative processes, e.g. angiogenesis, and are active in neoplastic states, including von Recklinghausen's disease (neurofibromatosis). Their involvement may have been underestimated in neuropathological studies, to date, by a reliance on staining techniques that are inadequate for identifying degranulated and therefore activated mast cells (4). More exacting histochemical and immunostaining procedures will help to fully realize the extent of their participation in physiological and pathological processes.

Effects of seven days of galactose feeding and aldose reductase inhibition on mast cells and vessel morphometry in rat sciatic nerve

The association between mast cells and vessel morphometry in sciatic nerve was examined after seven days in animals fed a diet of 40% d-galactose and compared to control rats and to galactose-fed animals treated with the aldose reductase inhibitor, Tolrestat. Electron microscopy revealed an increase in the total number of mast cells and the number of degranulated mast cells in galactose-fed animals (7.8 ± 2.9; 2.6 ± 2.9; mean ± SD) compared to controls (4.6 ± 2.1; degranulated mast cells were not seen in any control nerves) and Tolrestat-treated, galactose-fed animals (4.4 ± 2.5; 0.1 ± 0.4). Although no significant differences were noted in the numbers of vessels between the three groups, an index of vasoconstriction was significantly increased in the galactose-fed animals (0.115 ± 0.048; mean ± SD) compared to controls (0.068 ± 0.011) and Tolrestat-treated, galactose-fed animals (0.075 ± 0.020). These data suggest that mast cell degranulation is associated with the vascular constriction induced by seven days of galactose intoxication and that both may be prevented by inhibiting aldose reductase.

In utero galactose intoxication in animals.

To assess in utero effects of galactose, diets containing large amounts of the sugar have been fed to pregnant rats with examination of the fetuses and newborns. Galactose levels in fetal blood parallel those of the dam and amniotic fluid contains high concentrations of galactose and galactitol. In the offspring and in the fetuses cataracts, and a decrease in brain weight, protein content and DNA are observed. There is an elevated content of galactitol and decrease in myo-inositol in the brain of galactose-intoxicated newborns. Brain synaptosomes of young rats whose mothers were fed galactose show a reduced response in vitro to acetylcholine as evidenced by a decrease in incorporation of myo-inositol into phosphatidylinositol and an inappropriate change in phosphatidylinositol content. The data indicate that there can be significant effects in the offspring of pregnant rats fed a high galactose diet. The findings support the concept that in utero toxicity may occur in the human galactosemic fetus.

Mast cell degranulation and blood-nerve barrier permeability in rat sciatic nerve after 7 days of hyperglycemia

The association between hyperglycemia and altered blood-nerve barrier permeability was examined after 7 days of experimental diabetes. In nerves of rats fed a diet of 40% galactose, permeability to [14C]mannitol [13.43 +/- 2.47 x 10(-5) (SD) ml.s-1.g dry wt-1] and water content [3.43 +/- 0.24 (SD) mg/mg dry wt] were significantly increased compared with control (9.24 +/- 2.09 x 10(-5) ml.s-1.g dry wt-1 and 2.15 +/- 0.28 mg/mg dry wt) and streptozotocin-diabetic animals (8.43 +/- 2.94 x 10(-5) ml.s.-1.g dry wt-1 and 2.35 +/- 0.56 mg/mg dry wt). Electron microscopy revealed significant increases in the number of degranulating perivascular mast cells and in an index of vasoconstriction in galactose-treated rats (3.8 +/- 1.6 and 0.160 +/- 0.062, respectively) compared with control (0.5 +/- 0.8 and 0.072 +/- 0.017, respectively) and diabetic animals (1.4 +/- 1.7 and 0.083 +/- 0.033, respectively). The data are consistent with a role for mast cells in permeability changes occurring after only 7 days of galactose intoxication.

Tolrestat treatment prevents modification of the formalin test model of prolonged pain in hyperglycemic rats

This study examined the effects of hyperglycemia and treatment with the aldose reductase inhibitor, Tolrestat, on the pain behavior evoked by injection of formalin into the dorsum of a single hind paw. In control rats, injection of formalin (50 μl of a 5% solution) evoked two phases of flinching of the injected paw (phases 1 and 2), separated by a quiescent period. Four weeks of streptozotocin-induced diabetes or galactose intoxication did not alter the frequency of flinching during either of the active phases but significantly ( P < 0.001 and P < 0.05, respectively) enhanced flinch frequency during the quiescent period. Concurrent treatment with Tolrestat (50 mg/kg/day by gavage) during hyperglycemia prevented the accumulation of the polyol pathway metabolites sorbitol and fructose in the nerve and spinal cord of streptozotocin-diabetic rats and also significantly ( P < 0.05) reduced the enhanced flinching of diabetic rats during the quiescent period. These data demonstrate that hyperglycemia induces a period of Tolrestat-preventable hyperalgesia in a paradigm that is used to model persistent pain and suggest that exaggerated flux through aldose reductase may initiate changes in nociceptive pathways that could contribute to some of the pain states experienced by patients with diabetic neuropathy.

Inhibition of Macrophage Chemotaxis and Peripheral Nerve Regeneration in Normal and Hyperglycemic Rats by the Aldose Reductase Inhibitor Tolrestat

This study examined the effect of Tolrestat, an inhibitor of aldose reductase, on the regenerative capacity and macrophage chemotactic property of crush-injured sciatic nerve in normal and galactose-fed rats. Galactose intoxication reduced the incidence of regeneration but did not alter the regeneration distance or the injury-induced increase in vasoactive intestinal polypeptide content of dorsal root ganglia. Tolrestat improved the incidence of regeneration in galactose-fed rats but significantly (P < 0.05) reduced the distance of nerve regeneration in both control and galactose-fed rats. Galactose intoxication enhanced the ability of homogenates of nerve undergoing Wallerian degeneration to attract macrophages, whereas chemotaxis toward nerve homogenates from Tolrestat-treated rats was absent. Tolrestat, but not the structurally dissimilar aldose reductase inbibitors Ponalrestat and Sorbinil, exhibited a reversible, dose-dependent inhibition of macrophage chemotaxis induced by polyinosinic acid. These data suggest that exaggerated sugar metabolism by aldose reductase may restrict the ability of nerve to initiate regeneration but is not responsible for the reduced distance of nerve regeneration or attenuated increase in vasoactive intestinal polypeptide production that occur after crush injury of diabetic rats. Inhibition of macrophage responses to chemotactic signals by Tolrestat may impede regeneration and other reparative mechanisms.

Permeability and surface area of the blood-nerve barrier in galactose intoxication

The blood-nerve movement of a small molecular weight non-electrolyte was studied in control and galactose-fed rats by measuring the permeability-surface area (PSA) product of the blood-nerve interface to [ 14C]mannitol in sciatic nerve using an in vivo injection method. PSA products were measured after 9 to 11 months of feeding control rats a diet containing 0% galactose and galactose-intoxicated rats a diet containing 40% galactose. Nerves of the galactose-fed group were hydrated as reflected by a significant increase in nerve water content and wet weight to dry weight ratio (both P < 0.05). Compared to controls, PSA products were increased by 51% ( P < 0.01) in galactose-fed animals when referenced to nerve dry weight (13.59 ± 2.90 × 10 −5ml/s/g dry wt. versus 8.99 ± 1.59 × 10 −5ml/s/g dry wt.; mean ±S.D.; galactose vs. controls, respectively) or by 30% ( P < 0.001) when referenced to nerve length (± 0.43 × 10 −5ml/s/mm vs.1.87 ± 0.48 × 10 −5ml/s/mm) but not when referenced to nerve wet weight. It is suggested that in galactose intoxication, where endoneurial volume changes reflect increases in nerve water content, PSA products are best normalized to dry weight or length, which are not affected by volume changes. Normalized to dry weight, the blood-nerve barrier surface area (i.e. vessels and perineurium) was determined by morphometric methods to be increased by 34% in the galactose-intoxicated group. Although this increase is consistent with the observed increase in PSA product, most of the increased surface area occurs in the perineurium, which is believed to have much less of a role than endoneurial vessels in blood-nerve transfer of hydrophilic non-electrolytes. However, endoneurial vessel tight junctional ‘area’ was calculated with morphometric methods to be increased by 53% in galactose-fed rats ( P < 0.005). This increase,presumably due to endothelial cell proliferation, is sufficient to explain the increased PSA product.

Schwann Cell Injury is Attenuated by Aldose Reductase Inhibition in Galactose Intoxication

Four months of galactose intoxication induces a dose-dependent osmotic imbalance of the nerve microenvironment characterized by polyol, water, and electrolyte accumulation. Recently, dose-dependent cellular lesions have been described in the sciatic nerves of galactose-intoxicated rats. The present study was designed to demonstrate that the cell injury and endoneurial osmotic imbalance in galactose intoxication are dependent on the subsequent metabolism of galactose by the polyol pathway. Three groups of age-matched, female Sprague-Dawley rats were fed a control diet or diets containing complete micronutrient supplements with 40% galactose or 40% galactose and 0.04% Ponalrestat, an aldose reductase inhibitor (ARI). After 4 to 5 months, sciatic nerves were analyzed for polyol, water and endoneurial electrolyte content and processed for light and electron microscopic examination. Ponalrestat prevented myo-inositol depletion and accumulations of dulcitol, water and endoneurial fluid electrolytes. Axonal size-frequency histograms revealed that Ponalrestat attenuated the shift toward smaller fibers and the decrease in mean axonal diameter seen in untreated galactose-fed rats. Electron microscopic examination showed widespread reactive and degenerative changes in Schwann cells of galactose-intoxicated rats that culminated in cytoplasmic disintegration. Quantitative electron microscopy revealed that ARI treatment significantly reduced the incidence of abnormal Schwann cells. These observations indicate that the osmotic imbalance and cell injury seen in galactose intoxication are dependent on the metabolism of galactose by the polyol pathway.

Dose-dependent alterations in nerve polyols and (Na +,K +)-ATPase activity in galactose intoxication

Nerve polyol content and (Na +,K +)-adenosine triphosphatase (ATPase) activity of nerve homogenates were studied in a colony of rats fed diets containing either 0%, 10%, 20%, or 40% galactose for 4 months. Nerve water and dulcitol content exhibited dose-dependent increases, whereas nerve myo-inositol content declined with increasing dietary galactose. Homogenate (Na +,K +)-ATPase activity increased with increasing galactose consumption of up to 20% dietary intake and thereafter remained consistently elevated at twice the activity of 0% galactose-fed values. Nerves of rats fed 40% galactose were also examined at the light microscope level and showed evidence of both edema and myelin splitting. These data demonstrate that increased nerve water content, dulcitol accumulation, and myo-inositol depletion parallel the previously reported dose-related increase of endoneurial fluid sodium and chloride in nerves of galactose-fed rats and suggest that elevated nerve homogenate (Na +,K +)-ATPase activity may be related to one or more of these consequences of exaggerated polyol pathway flux.

Cellular Pathology of the Nerve Microenvironment in Galactose Intoxication

The effect of chronic hyperglycemia and polyol pathway activation on the Schwann cell has not been resolved although injury to this cell has long been suspected in diabetic neuropathy. Hyperglycemia, resulting from galactose intoxication of four months duration, induces dose-dependent accumulations of endoneurial fluid sodium and chloride that are linked to polyol pathway activity and associated with dose-dependent increases in sciatic nerve water content, endoneurial fluid pressure and (Na+, K+)-ATPase activity. In order to understand the impact of these changes on the nerve microenvironment, cellular elements of the endoneurium were quantitatively and qualitatively assessed in rats receiving 0%, 10%, 20% or 40% galactose diets. After four months of galactose intoxication, dose-dependent changes in the size distribution of myelinated nerve fibers were apparent. A shift in size-frequency histograms of galactose-intoxicated animals towards smaller fibers was accompanied by a decrease in axon diameter and the volume fraction ratio of axon to myelinated nerve fibers. In the sciatic nerve of all 40% galactose-fed rats examined by electron microscopy, Schwann cells of myelinated fibers showed both reactive and degenerative changes. Demyelination was preceded by splitting at the intraperiod line. Remyelination was identified by axons with disproportionately thin myelin sheaths. Axonal dystrophy and degeneration were infrequently seen, but there was axonal regeneration. Dose-dependent increases in mast cell number were observed with degranulation apparent in rats receiving 20% and 40% galactose. Endothelial cell number and basal lamina thickness were increased in the endoneurial vessels of galactose-intoxicated rats. Increased cytoplasmic area and degenerative changes in pericytes were also noted. These observations indicate that significant morphologic changes accompany the hyperosmotic imbalance resulting from galactose intoxication of four months duration. Schwann cell injury and demyelination are present in a disorder linked to polyol metabolism since aldose reductase, the anabolic enzyme of the polyol pathway, is localized to this myelin-forming cell.

Dose-dependence of endoneurial fluid sodium and chloride accumulation in galactose intoxication

Endoneurial edema in galactose neuropathy was studied in a colony of Sprague-Dawley rats fed diets containing 0%, 10%, 20% or 40% d-galactose for approx. 200 days. Endoneurial fluid was analyzed by X-ray microanalysis for electrolyte concentration, by microgravimetry of whole nerve segments for water content, by measurement of endoneurial fluid pressure and by morphometry in transverse sections of nerve. Galactose intoxication resulted in dose-dependent increases in endoneurial fluid sodium and chloride that were directly associated with increases in nerve water content and endoneurial fluid pressure. The presence of edema and its dose-dependence was also confirmed by morphometric analysis of sciatic nerves at the light microscopic level. The data demonstrate that electrolyte-induced osmotic imbalances in endoneurial fluid are dependent on the amount of galactose ingested and suggest that the dose-related accumulation of sodium and chloride in endoneurial fluid contributes substantially to the pathogenesis of galactose neuropathy.

Galactose neuropathy

Galactose neuropathy is characterized by progressive endoneurial edema manifested by a gradual increase in endoneurial fluid pressure. Edema accumulates via a unique mechanism of osmotic force generated by products of the polyol pathway, synthesized within the endoneurial compartment. This paper presents morphologic findings showing firstly, that blood nerve barrier permeability to horseradish peroxidase complexes appears unchanged and secondly, peripheral nerve edema in this condition is restricted to extraganglionic endoneurium sparing the spinal ganglia and adjacent roots. Thirdly, mast cells accumulated in significant numbers and electron microscopy revealed degranulation. There was no evidence of edema in Schwann cell cytoplasm, the putative site of galactitol accumulation via the sorbitol pathway. These findings are discussed with respect to diabetic neuropathy for which galactose intoxication is a useful experimental model.

GALACTOSE INTOXICATION. PATHOLOGIC STUDY IN THE CHICK.

Des poussins nourris avec une ration contenant plus de 10% de galactose developpent des tremblements, des convulsions et meurent. Une degeneration des neurons et de l'oedema peri-cellulaire se presente dans la zone des ganglions basals, dans la zone medullaire et dans les lobes optiques.