Reversibility of neurofilamentous inclusion formation following repeated sublethal intracisternal inoculums of AlCl3 in New Zealand white rabbits.

In this report, we describe the clinical, topographical and immunohistochemical characteristics of neurofilament (NF) inclusion formation induced by the intracisternal inoculation of young adult New Zealand white rabbits at 28-day intervals with 100 micrograms AlCl3 over the course of 267 days. The ability to recover following cessation of aluminum exposure has also been assessed. The extent of neurofilamentous inclusion formation was proportionate to the cumulative amount of AlCl3 inoculated and initially consisted of fusiform axonal distention in the ventral spinal cord at day 51 following the initial inoculum. Spinal motor neuron perikaryal inclusions and discrete axonal spheroids were observed at day 107 and supraspinal neurofilamentous pathology by day 156. Perikaryal inclusions were immunoreactive to antibodies recognizing both poorly phosphorylated (SMI 32) and more highly phosphorylated high molecular weight NF (NFH). In contrast, axonal spheroids were intensely immunoreactive at all stages with antibodies recognizing highly phosphorylated NFH and an age-dependent NFH phosphorylation state (SMI 34) with only faint SMI 32 immunoreactivity. Immunoreactivity to an antibody recognizing ubiquitin-protein conjugates did not appear until day 156, whereas inclusions were not immunoreactive to antibodies recognizing either phosphatase-dependent or -independent microtubule-associated protein tau at any stage. Upon withdrawal from further AlCl3 exposure after intervals of 51, 107 or 156 days following the initial inoculum, clinical recovery ensued in all rabbits. In all but the most severely affected rabbits, perikaryal neurofilamentous inclusions resolved. However, axonal spheroids continued to be prominent. These studies demonstrate that the repetitive intracisternal inoculation of AlCl3 in New Zealand white rabbits induces a reversible process of neurofilamentous inclusion formation that preferentially affects motor neurons, and in which recovery will occur in those inclusions containing an admixture of both poorly and highly phosphorylated NFH.

200 kDa and 160 kDa neurofilament protein phosphatase resistance following in vivo aluminum chloride exposure.

We have used time-course dephosphorylation experiments and two dimensional isoelectric focusing to assess the phosphorylation state of neurofilament (NF) proteins following the intracisternal inoculation of AlCl3. Littermates of New Zealand white rabbits, age 5-6 weeks, were inoculated with either 1000, 750, 500, 250 or 100 micrograms AlCl3 in 0.9% NaCl or 0.9% NaCl alone, killed 48 hours later and the NF-enriched cytoskeletal fraction isolated from the spinal cord. Neurofilamentous inclusions did not occur following inoculums of 100 or 250 micrograms AlCl3, but thereafter developed in increasing quantities in a dosage-dependent manner. Incubation of the NF-enriched fraction with E. Coli. alkaline phosphatase (enzyme: substrate 1:50) induced a replacement of the highly phosphorylated 200 kDa isoform of NFH with a more poorly phosphorylated 170 kDa isoform, confirmed by immunoblot analysis. This reaction was complete within 20 minutes with NF derived from NaCl, 100 or 250 micrograms AlCl3 inoculated rabbits and within 30 minutes for 500 micrograms AlCl3 inoculums. However, residual highly phosphorylated NFH isoforms persisted at 60 minutes for 750 micrograms inoculums and 90 minutes for that derived from 1000 micrograms AlCl3 inoculums. A similar inhibition of phosphatase activity was observed for NFM. Following two dimensional electrophoresis of the NF-enriched isolate, no alteration in the net phosphorylation state of individual NF subunit proteins was observed--regardless of the inoculum. These results demonstrate a dose-dependent induction of neurofilamentous inclusions in spinal motor neurons following intracisternal AlCl3 inoculation accompanied by increasing phosphatase resistance without a demonstrable alteration in NF net phosphorylation state.

Effects of alcohol withdrawal on responses of platelets from alcoholics--a study using platelet-rich plasma from blood anticoagulated with D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (FPRCH2Cl).

Platelet responses stimulated by a range of concentrations of ADP or collagen were studied in platelet-rich plasma (PRP) from alcoholics 24-36 h and 6 days after cessation of drinking, and in PRP from age- and sex-matched controls. The studies were done using plasma from blood anticoagulated with the specific thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (FPRCH2Cl, PPACK); the use of this compound permits the study of platelet responses in plasma at physiological concentrations of ionized calcium. Responses of platelets to ADP (primary aggregation) and collagen (aggregation, secretion of [14C]serotonin from prelabelled platelets, and thromboxane formation) were lower in alcoholics 24-36 h after withdrawal of alcohol compared with controls. This inhibition of platelet function was not due to the presence of alcohol in the blood of the alcoholics. Aggregation in response to ADP did not change during the withdrawal period studied, while collagen-induced aggregation and secretion increased significantly and collagen-induced thromboxane formation tended to increase towards control values. The reduced platelet responses observed in alcoholics and the different rates of "recovery" of different pathways of aggregation towards control values must be due to alterations either in the platelets themselves and/or in the plasma brought about by the chronic presence of ethanol, and its withdrawal.

Platelet responses to platelet-activating factor are inhibited in alcoholics undergoing alcohol withdrawal.

Platelet aggregation, secretion of serotonin, and formation of thromboxane B2 induced by platelet-activating factor (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) were studied in plasma containing physiological concentrations of ionized calcium in eight alcoholics after cessation of heavy drinking. Responses of platelets of four nonalcoholic volunteers, matched with a subgroup of the alcoholics by age and sex, were also investigated. Aggregation of platelets from alcoholics was significantly less throughout the 6-day detoxification period compared with controls. Secretion of serotonin (5-hydroxy-tryptamine) was negligible and the production of thromboxane B2 was not detectable. Decreased platelet aggregability in response to aggregating agents, including platelet-activating factor, may be important in the development of hemorrhagic complications in alcoholics.

In vitro effects of ethanol on rabbit platelet aggregation, secretion of granule contents, and cyclic AMP levels in the presence of prostacyclin.

Ethanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin-treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.