Use of lumbar laminoplasty vs. laminotomy for transection of the filum terminale does not affect early complication rates or postoperative course.

INTRODUCTION: Various techniques are used for spinal cord untethering. The purpose of this study was to compare patient characteristics, postoperative course, and early complications after laminotomy vs. laminoplasty for transection of the filum terminale for tethered cord release. METHODS: Retrospective analysis of clinical and magnetic resonance imaging data was undertaken for all patients (<18 years) who underwent tethered cord release by transection of the filum terminale at Oregon Health & Science University, Doernbecher Children's Hospital, from 2000 to 2011. RESULTS: Data from two hundred and forty-eight patients were analyzed. Mean age was 5.2 years (range 0.3 to 16.8 years). Access to the thecal space during surgery was achieved using laminotomy or laminoplasty in 82 (33.1 %) and 166 (66.9 %) patients, respectively. Laminoplasty patients were significantly younger than laminotomy patients (3.2 vs. 9.3 years, p<0.0001); other clinical and radiographic characteristics were similar between the groups. Nine patients (3.6 %) experienced early complications, including cerebrospinal fluid leak (n=2), suprafascial infection requiring surgical management and intravenous (IV) antibiotics (n=3) or IV antibiotics alone (n=1), a small area of peri-incisional cutaneous necrosis (n=1), perioperative seizures (n=1), and mild, transient malignant hyperthermia (n=1). There was no difference in the number of early complications between the two groups. Univariate and multivariate analyses revealed no significant risk factor for postoperative complication associated with technique. As judged by caregivers, independent of surgical technique, 97 % of patients improved after surgery. CONCLUSION: There was no difference in complication risk when performing transection of the filum terminale for tethered cord release using laminotomy or laminoplasty.

Expression and cellular localization of the classical progesterone receptor in healthy and amyotrophic lateral sclerosis affected spinal cord.

BACKGROUND AND PURPOSE: Previous studies have suggested that elevated progesterone levels are associated with a slower disease course in amyotrophic lateral sclerosis (ALS). Given that the effects of progesterone are mediated in part by the classical progesterone receptor (PR), the expression and cellular localization of the A and B isoforms (PR-A and PR-B, respectively) of the PR in control (neuropathologically normal) and ALS-affected spinal cord (SC) were examined. METHODS: Semi-quantitative RT-PCR, immunohistochemistry and immunofluorescence analyses of the cervical and lumbar SC of post-mortem ALS patients (n = 19) and control subjects (n = 10) were performed. Primers and antibodies used allowed the detection of both PR-A and PR-B isoforms together (PR-A+B) or PR-B isoform alone. RESULTS: Lumbar PR-A+B and cervical PR-B mRNA expression were significantly higher in ALS than controls. In both ALS and controls, PR-A+B immunoreactivity (IR) was occasionally detected in motor neurons. In contrast, PR-A+B IR was prominent in axonal processes and vessels. This was more evident in nerve roots and large arteries in ALS compared with controls. Colocalization of PR-A+B with markers of neurons, axonal processes and vascular endothelium was also observed. CONCLUSIONS: Evidence that both PR-A and PR-B isoforms are expressed in the human SC is provided, with some regional variation in isoform expression between ALS and controls. The IR was more prominent in nerve roots and large arteries in ALS, suggesting a potential role in the degenerative process.

Regional spread pattern predicts survival in patients with sporadic amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Sporadic amyotrophic lateral sclerosis (sALS) is a disease with a focal clinical onset and contiguous spread. We examined patterns of disease spread following symptoms onset in sALS and whether the pattern of spread predicted survival. METHODS: Review of medical records (2003-2009) at London Ontario and Buenos Aires clinic cohorts retrieved 318 patients with sporadic sALS. According to patient self-report, we determined eight spread patterns: rostro-caudal, caudo-rostral, crossed, circular, superior interposed, middle interposed, inferior interposed and isolated. The variables studied were as follows: age, gender, sALS phenotypes, time from onset to diagnosis and time and direction of the spreading to the first region. Survival from symptoms onset was analysed by Kaplan-Meier, Tarone-Ware and Cox proportional hazards methods. RESULTS: The direction of first spread was horizontal in 33%, rostral to caudal in 32% and caudal to rostral in 21%, whereas spread to remote regions was observed in 14% of patients. Survival curves and 3- and 5-year survival rates favoured patients with an isolated and caudo-rostral pattern of spread compared to patients progressing to distant regions without involvement in the intervening region, or 'superior and inferior interposed patterns' (Tarone-Ware P = 0.001, χ(2) = 0.002 and χ(2) = 0.006, respectively). Factors affecting survival were gender, time to diagnosis, flail arm phenotype and age at diagnosis. CONCLUSIONS: We have provided evidence that not all spread in ALS is contiguous and that the nature of symptom progression influences survival. Patients with sALS with 'interposed patterns' had a worse prognosis, whereas patients with caudo-rostral pattern fared better than the rest.

Nitric oxide synthase expression in cervical spinal cord in sporadic amyotrophic lateral sclerosis.

Nitration of neurofilament (NF) has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Evidence of such includes elevated 3-nitrotyrosine levels in spinal cord tissue and localized nitrotyrosine immunoreactivity with neurofilamentous aggregates in cortical and spinal motor neurons. To determine if neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) are the sources of nitric oxide in sporadic ALS (sALS), particularly through over-expression of the enzyme, steady-state mRNA levels of these isoforms were studied by in situ hybridization. Paraffin-embedded, archival cervical spinal cord tissues from 7 sALS and 6 control cases were used. 35S-labeled riboprobes were generated from partial cDNAs. Immunohistochemistry was utilized to confirm results of iNOS hybridization. We observed that nNOS mRNA was constitutively expressed in cervical spinal motor neurons. However, iNOS mRNA and iNOS immunoreactivity was not observed in ALS or control motor neurons. Our observations suggest that the source of nitric oxide is the endogenous nNOS. Together with the results from other immunohistochemical studies, we further hypothesize a possible role of translational deregulation of nNOS in sALS.

Proximal sciatic axotomy does not inhibit the induction of neurofilamentous inclusions following intracisternal aluminum chloride exposure.

We have previously demonstrated an acute, dose-dependent suppression of low molecular weight neurofilaments (NFL) and intermediate molecular weight neurofilaments (NFM) steady state mRNA levels while sparing those of high molecular weight (NFH) mRNA 48 hours (h) following the intracisternal inoculation of AlCl3 in young adult New Zealand white rabbits. To determine whether this alteration in NF steady state mRNA stoichiometry is a necessary prerequisite to the induction of neurofilamentous inclusions, we examined the response of spinal motor neurons to aluminum exposure in vivo following axotomy. Forty-eight h following a complete transection of the proximal sciatic nerve, rabbits were inoculated intracisternally with either 1000 microg AlCl3 in 100 microl 0.9% NaCl or 0.9% NaCl alone. Rabbits were killed at either 48 or 120 h post-inoculation, and the extent of neurofilamentous inclusion formation quantified in both the cervical and the lumbosacral cord. Following the axotomy, rabbits developed an ipsilateral hind-limb paralysis. In spinal motor neurons ipsilateral to the axotomy, chromatolytic changes were observed and both NFH and NFM mRNA levels were significantly reduced (p<0.001). At 48 h post-AlCl3 inoculation, 29% of motor neurons contralateral to the axotomy demonstrated inclusions, whereas 43% of ipsilateral motor neurons demonstrated inclusions (Fisher's test, two tailed, p = 0.0196). At 120 h post-axotomy 75% and 83%, respectively, of neurons were involved (p = 0.0212). Neurofilamentous inclusions did not form in NaCl-inoculated rabbits. These observations indicate that an altered stoichiometry of NF mRNA steady levels, with a relative overexpression of NFH mRNA, is not critical to the induction of neurofilamentous inclusions following AlCl3 exposure.

Characterization of neuronal intermediate filament protein expression in cervical spinal motor neurons in sporadic amyotrophic lateral sclerosis (ALS).

Because transgenic mice expressing an altered stoichiometry of neurofilament proteins develop a motor neuron degeneration associated with neurofilamentous aggregate formation similar to that found in amyotrophic lateral sclerosis (ALS), we studied the expression of intermediate filament proteins in sporadic ALS. Archival cervical spinal cord paraffin-embedded sections from 11 disease and 11 control cases were studied by either in situ hybridization using 35S-labeled riboprobes or immunohistochemically using specific antibodies for the individual neurofilament subunit proteins, alpha-internexin, nestin, peripherin, vimentin, beta-actin, or Talpha1-tubulin. Median NFL, alpha-internexin, and peripherin steady-state mRNA levels were significantly reduced in the lateral motor neuron cell column (p < 0.05) of ALS cases, while neither NFM nor NFH mRNA levels were altered. ALS cases demonstrated an elevation of beta-actin mRNA levels (p < 0.01) with no increase in Talpha1-tubulin mRNA levels. No motor neuronal expression of nestin or vimentin was observed. Ubiquitin-immunoreactive perikaryal aggregates were immunoreactive for NFH or beta-actin, but not for peripherin, alpha-internexin, vimentin, or nestin. In contrast, neuroaxonal spheroids were strongly immunoreactive for NFH and peripherin, but not for beta-actin, alpha-internexin, vimentin, or nestin. These findings suggest that the stoichiometry of cytoskeletal protein expression in ALS spinal motor neurons is significantly altered in a pattern conducive to the formation of neurofilamentous aggregates.

Case of the month: June 1997--a 42 year old man with left facial weakness.

A 42 yr old male presented with left facial weakness. MRI showed lesions affecting the distal seventh nerve and third division of the trigeminal nerve. The seventh nerve was biopsied and showed a malignant epithelioid schwannoma. The patient underwent extensive resection followed by irradiation. This is one of very few examples of intracranial malignant peripheral nerve sheath tumors and the first reported example of an intracranial malignant epithelioid schwannoma. The literature is reviewed and completeness of resection appears to be the most pertinent prognostic factor.

Simplifying the approach: what can we do?

Although it is desirable to simplify the diagnosis of amyotrophic lateral sclerosis (ALS), it is not obvious how to achieve this. Although a simplified electrophysiologic approach can be easily performed, interpretation of the results in a complex differential diagnosis is far from simple. Genetic screening may potentially be useful but is still in its infancy, even in familial ALS. Spasticity scales are most informative when greater degrees of spasticity are present, but usually these cases can also be identified clinically. It is difficult to envisage simplifying the scales because bedside rating scales are inherently not good at the mild end of the spectrum. Although MRI may prove to be useful in simplifying the diagnostic procedure, at present the numbers of patients studied are too small and the findings not sufficiently specific. Among the major issues that must be decided are whether any of the newer diagnostic techniques are sufficiently reliable, sensitive, and specific to allow any further simplification, and whether paraclinical tests are acceptable surrogates for clinical phenomena. The diagnostic approach of moving a possible diagnosis of ALS into the category of probable ALS earlier by giving more weight to electrophysiologic findings appears to be the only option, together with a more rigorous approach to excluding disorders known to mimic ALS. The problem lies less in the specialist reaching a correct diagnosis than at the level of initial evaluation, where the suspicion of ALS may be raised in only 27-40% of cases.

Cognitive impairment in sporadic ALS: a pathologic continuum underlying a multisystem disorder.

BACKGROUND: Traditionally considered a motor neuron-selective disorder, the clinical manifestations of ALS can include a frontotemporal dementia. Although the pathologic substrate of cognitive impairment remains to be defined, the presence of ubiquitin-immunoreactive (Ub+) intraneuronal inclusions in cortical regions has been suggested to constitute a pathologic marker of this process. METHODS: The authors compared the neuropathological features of four cognitively impaired patients with ALS, four cognitively intact patients with ALS, and four neurologically normal patients. The extent and load of Ub+ neuronal inclusions, Ub+ dystrophic neurites, and superficial linear spongiosis (SLS) was determined among a number of cortical, hippocampal, and subcortical regions. RESULTS: Although Ub+, alpha-synuclein-negative, and tau-negative neuronal inclusions were observed in both cognitively impaired and cognitively intact patients with ALS, their density and extent was greater among the former, with the difference greatest in the cingulate gyrus. Ub+ neurites were observed in a similar distribution. Only the presence of SLS, affecting the first and second cortical layers, reliably distinguished between the cognitively impaired and cognitively intact ALS subpopulations. In three of four cognitively impaired patients with ALS, SLS was associated with transcortical microglial activation, in the absence of detectable differences in astrocytosis, density of calbindin or parvalbumin neurons, or optical density of synaptophysin and SNAP-25. CONCLUSIONS: Although intraneuronal Ub+ inclusions and dystrophic neurites are observed in both ALS subpopulations, the presence of cognitive impairment was associated with a greater distribution and load of both neuropathologic features, suggesting a disease continuum. Moreover, cognitive impairment was uniformly associated with superficial linear spongiosis, a pathologic feature common to several forms of frontotemporal dementia.

A prospective study of cognitive impairment in ALS.

OBJECTIVE: To characterize prospectively the cognitive profile in ALS. METHODS: Clinically definite ALS patients (11 men, 2 women), age 39.9 to 74.0 years (mean age, 54.2 +/- 9.6 years; mean disease duration, 21.1 +/- 10.5 months) underwent neuropsychologic, language, and speech testing followed by MR 1H spectroscopy (4 T). Five spousal control subjects completed an identical protocol. Eight ALS patients participated in follow-up studies at a 6-month interval. RESULTS: Relative to control subjects, ALS patients showed mild impairment in word generation, recognition memory (faces), and motor-free visual perception. Bulbar-onset patients showed greater impairment in a number of measures (working memory, problem solving/cognitive flexibility, visual perception, and recognition memory for words and faces), and cognitive impairment appeared more progressive over time. ALS patients demonstrated anomia on a confrontation naming test, with no significant problems following commands or repeating. Speech motor performance scores and intelligibility scores were not significantly different. No significant declines in forced vital capacity, forced expiratory volume, or peak expiratory flow rates were observed. Although normal at initial testing (T1), MR 1H spectroscopy demonstrated a reduction of the N-acetylaspartate/creatine (NAA/Cr) ratio in the nondominant precentral motor strip across the two testing intervals. In contrast, the NAA/Cr ratio obtained from the anterior cingulate gyrus at T1 was already reduced in bulbar-onset patients (p < 0.001), whereas no deficits were observed in limb-onset individuals in the same region. CONCLUSIONS: Bulbar-onset ALS patients with cognitive impairments and neuronal loss in the anterior cingulate gyrus subsequently developed more profound neuropsychological dysfunction whereas both language and speech capabilities remained relatively preserved. Of note, the absence of bulbar signs did not predict an absence of cognitive decline.

Sleep-disordered breathing in amyotrophic lateral sclerosis.

OBJECTIVE: The purpose of this study was to assess sleep and breathing in patients with amyotrophic lateral sclerosis (ALS) with bulbar muscle involvement. DESIGN: Prospective, controlled study of sleep and breathing measured during polysomnography. SETTING: University teaching hospital and referral center. PATIENTS: Patients with definite ALS and healthy age-matched control subjects. INTERVENTIONS: Eighteen ALS patients and 10 age-matched control subjects underwent one night of polysomnography. Thirteen patients with ALS were studied for a second night. RESULTS: The ALS patients had more arousals per hour (p = 0.008), more stage 1 sleep (p = 0.01), and a shorter total sleep time (TST) (279 +/- 69 vs 331.4 +/- 55.9 min, mean +/- SD, p = 0.04) than the control subjects. The ALS patients had mild sleep-disordered breathing with a greater apnea/hypopnea index (AHI) than the control subjects (p = 0.005). On the second night of polysomnography, there was an increase in TST (p = 0.003) and rapid eye movement (REM) sleep (p = 0.009), an improvement in sleep efficiency (p = 0.02), and less stage 1 sleep (p = 0.04). Eight ALS patients had sleep-disordered breathing consisting of periods of hypoventilation, predominantly during REM sleep. CONCLUSIONS: Sleep-disordered breathing occurs in patients with ALS and is similar to patients without ALS with respiratory muscle weakness. No obstructive sleep apnea was observed. One potential reason for its absence might be the inability of patients with respiratory muscle weakness to generate an inspiratory pressure greater than the upper airway closing pressure. This hypothesis should be addressed in future studies.

A morphological analysis of the motor neuron degeneration and microglial reaction in acute and chronic in vivo aluminum chloride neurotoxicity.

The monthly intracisternal inoculation of aluminum chloride (AlCl3) to young adult New Zealand white rabbits induces motor neuron degeneration marked by intraneuronal neurofilamentous aggregates similar to that observed in amyotrophic lateral sclerosis (ALS). However, in contrast to ALS, this process occurs in the experimental paradigm in the absence of a glial response. In addition, whereas ALS is a fatal disorder, the cessation of aluminum exposure leads to both clinical and neuropathological recovery. Because microglia can influence neuronal regeneration, we have examined the effect of both acute and chronic aluminum exposure on microglial activation in vivo. We have studied microglial morphology in young adult New Zealand white rabbits receiving either single (1000 microg) or repeated sublethal (100 microg monthly) intracisternal inoculums of AlCl3. In addition, rabbits receiving 1000 microg AlCl3 inoculums were studied following an unilateral sciatic axotomy 48 h prior to the AlCl3 exposure. Our studies demonstrate that microglial activation in vivo is inhibited by AlCl3 exposure, and that a correlation exists between the extent of microglia suppression and the potential for recovery. This suggests that microglial activation is an important determinant of neuronal injury.

Aluminum neurotoxicity: an experimental approach to the induction of neurofilamentous inclusions.

Acute or chronic aluminum neurotoxicity experiments in the rabbit suggest that aluminum can induce phosphorylation of neurofilamentous proteins. This may result in abnormal resistance to degradation or transport of neurofilament protein and so to the accumulation of neurofilaments in abnormal cells. The possible importance of this process in ALS is considered in relation to the neurofilamentous abnormalities characteristic of intraneuronal inclusions in ALS and in other neurodegenerative disorders.

Neurofilament metabolism in sporadic amyotrophic lateral sclerosis.

Although the role of intraneuronal neurofilamentous aggregates in the pathogenesis of ALS is unknown, their presence forms a key neuropathological hallmark of the disease process. Conversely, the experimental induction of neurofilamentous aggregates in either neurotoxic or transgenic mice gives rise to motor system degeneration. To determine whether alterations in the physiochemical properties of NF are present in sporadic ALS, we purified NF subunit proteins from cervical spinal cord of ALS and age-matched control patients. The cytoskeleton-enriched, Triton X-100 insoluble fraction was further separated into individual NF subunits using hydroxyapatite HPLC. We observed no differences between control and ALS in the characteristics of NFH, including migration patterns on 2D-IEF, sensitivity to E. coli, alkaline phosphatase mediated dephosphorylation, peptide mapping, or proteolysis (calpain, calpain/calmodulin mediated, phosphorylated or dephosphorylated NFH). NFL showed no differences in 2D-IEF migration patterns, peptide mapping, or the extent of NFL nitrotyrosine immunoreactivity in either the Triton soluble or insoluble fractions. The latter observation demonstrated that NFL nitration is a ubiquitous occurrence in neurons and suggests that NFL might function as a sink for free reactive nitrating species. In contrast to the lack of differences in the post-translational processing of NF in ALS, we did observe a selective suppression of NFL steady state mRNA levels in the limb innervating lateral motor neuron column of ALS. This occurred in the absence of modifications in NFH, NFM or neuronal nitric oxide synthase (Type I NOS; nNOS) steady state mRNA levels. Coupled with previous observations of nNOS immunoreactivity co-localizing with NF aggregates in ALS motor neurons, this suggests activation of the nNOS enzyme complex in ALS, which would be predicted to contribute directly to the generation of reactive nitrating species. Given this, the isolated suppression of NFL steady state mRNA levels in ALS may indicate that ALS motor neurons are at an intrinsic deficit in the ability to buffer free reactive nitrating species.

Percutaneous gastrojejunostomy in amyotrophic lateral sclerosis.

We have performed a retrospective review of the use of a percutaneous gastrojejunostomy in patients with amyotrophic lateral sclerosis (ALS). Forty-one patients with initial bulbar manifestations of ALS and 32 patients with initial limb manifestations underwent a percutaneous gastrojejunostomy under fluoroscopic control using the Rankin gastrojejunostomy tube. Survival characteristics were compared with 86 bulbar onsetting and 207 limb onsetting ALS patients who did not require nutritional support. The 30-day mortality rate was 9.6% (respiratory death in three bulbar onsetting patients and four limb onsetting patients) and the 30 day morbidity rate was 4.1% (one operative site infection and intraperitoneal leakage in two patients). The most frequent long-term complication was the requirement for tube changing (blockage in six; dislodgment in two). Gastric reflux was not described amongst the treated patients. Overall survivorship (symptom onset to death) was less in the bulbar onsetting patients receiving a gastrojejunostomy tube than in the control population (median survival 22.0 vs. 33.7 months, respectively, P=0.005). As a group, the median survivorship for limb onsetting patients was not different for those receiving a gastrojejunostomy than for those who did not. However, a significant reduction in survival was observed in limb onsetting patients receiving a gastrojejunostomy early in the course of their disease (P=0.001) compared to those with a longer duration prior to the procedure. This was not observed in the bulbar onsetting patients. In both patient populations, no relationship was observed between survival post-gastrojejunostomy and the severity of pulmonary involvement at the time of the intervention, serum chloride, or age at onset. These studies demonstrate that a percutaneous gastrojejunostomy is a well-tolerated and safe alternative technique for enteral nutritional support in ALS patients. It also offers the advantage of not requiring either a general anaesthetic at the time of the procedure or instrumentation through the oropharynx. We have also observed that limb onsetting patients requiring a gastrojejunostomy early in the course of their illness are in a distinctive, less favorable, prognostic group.

Spinal motor neuron neuroaxonal spheroids in chronic aluminum neurotoxicity contain phosphatase-resistant high molecular weight neurofilament (NFH).

It has previously been shown that a single intracisternal inoculum of AlCl3 in young adult New Zealand white rabbits will induce a dose-dependent phosphatase resistance of high molecular weight neurofilament protein (NFH) that is proportionate to the extent of neurofilamentous inclusion formation (Strong and Jakowec, 1994). To determine if the potential for dissolution of aluminum-induced neurofilamentous inclusions was dependent on the degree of NFH phosphatase resistance, we have examined NFH phosphatase sensitivity in a reversible chronic model of aluminum neurotoxicity. Rabbits receiving repeated intracisternal inoculums of 100 microgram AlCl3 at 28 day intervals until day 267 develop spinal motor neuron perikaryal and neuroaxonal neurofilamentous aggregates in a stereotypic, dose-dependent fashion. In the rabbits receiving inoculums until day 156 with survival until day 267 without further aluminum exposure, neuroaxonal spheroids remained prominent while perikaryal inclusions largely resolved. Immunoreactivity to a monoclonal antibody recognizing phosphorylated NFH (SMI 31) was abolished in perikaryal aggregates at each time interval by dephosphorylation with bovine alkaline phosphatase. However, neuroaxonal spheroids maintained their immunoreactivity. Using time-course dephosphorylation studies of spinal cord homogenates, we observed a significant reduction in the rate of dephosphorylation of NFH following 267 days of AlCl3 exposure (P < 0.05). These observations suggest that neuroaxonal spheroids contain phosphatase-resistant NFH isoforms and that the potential for resolution of intraneuronal neurofilamentous inclusions correlates with the susceptibility of NF within these inclusions to enzymatic dephosphorylation.

Potentiation in the neurotoxic induction of experimental chronic neurodegenerative disorders: N-butyl benzenesulfonamide and aluminum chloride.

Repeated monthly intracisternal inoculations of N-butyl benzenesulfonamide induced a chronic, slowly progressive myelopathy in young adult New Zealand white rabbits that was manifested by hyperreflexia, spasticity, hypertonia, gait impairment and altered tonic immobility responses. The neuropathological features consisted of scattered neuroaxonal spheroids, fusiform distention of the intramedullary portions of the spinal cord ventral roots and, as defined by microtubule-associated protein-2 (MAP 2) immunoreactivity, an initial distention and subsequent loss of dendritic processes in neurons of the nucleus motoris lateralis with the perikaryon of these cells remaining intact. A similar chronic progressive myelopathy was induced by repeated low dose intracisternal inoculations of aluminum chloride in New Zealand white rabbits. However, the neuropathological changes were more extensive and consisted of dendritic, axonal and perikaryal inclusions of phosphorylated and nonphosphorylated neurofilament localized to spinal motor neurons in the nucleus motoris medialis, substantia grisea intermedia and select brainstem nuclei with only minimal involvement of the nucleus motoris lateralis. The co-administration of these two neurotoxins over the course of 8 months induced striking behavioral changes as well as a fulminant myelopathy. This was accompanied by a loss of neuronal perikarya in the nucleus motoris accompanied by a loss of neuronal perikarya in the nucleus motoris lateralis and topographically extensive neocortical neurofilamentous degeneration. These features suggest that potentiation occurs when the two toxins are co-administered, a view supported by an estimation of the co-neurotoxicity coefficient (CNC greater than 1). Our results have implications for understanding human neurodegenerative disorders in which potentiation of insults may occur, producing a clinical and neuropathological disease state not expected from either agent alone.

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.

Enhanced ex vivo cosedimentation of high molecular weight neurofilament protein (NFH) with microtubules following in vivo aluminum chloride exposure: inhibition of dephosphorylation-dependent dissociation.

We have investigated the effect of acute in vivo aluminum exposure on the subsequent ex vivo cross-linking of the high molecular weight neurofilament protein (NFH) with polymerized microtubules. Young adult female New Zealand white rabbits were inoculated intracisternally with 1000 micrograms of AlCl3 in 0.9% NaCl or with 0.9% NaCl alone, and killed 48 hours later. Following isolation of a cytoskeletal-enriched protein fraction from the cervical spinal cord, NFH was purified by either electroelution or column chromatography. Tubulin was isolated from New Zealand white rabbit brains by repeated temperature-dependent polymerization and depolymerization, purified over phosphocellulose, and cosedimented with either phosphorylated or dephosphorylated NFH. Following incubation for 30 minutes at 32 degrees C with tubulin in the presence of 20 microM Taxol, 1.0 mM MgCl2 and 1.0 mM GTP, the insoluble pellet containing NFH/microtubules was isolated. Both the pellet and supernatent were fractionated by SDS.PAGE and the amount of NFH present quantified by transmission densitometry following silver-staining. Results were identical regardless of the technique utilized for the purification of NFH. Control NFH preferentially cosedimented with microtubules when in the fully phosphorylated isoform, but remained in the soluble fraction following dephosphorylation. Phosphorylated NFH derived from AlCl3-inoculated rabbits demonstrated similar binding characteristics to control NFH, but following exhaustive dephosphorylation, exhibited a 4.5 fold induction of NFH/microtubule binding (p = 0.0314). Incubating dephosphorylated control NFH with microtubules in the presence of increasing concentrations of AlCl3 failed to induce similar cosedimentation. These experiments suggest that phosphorylation promotes NFH cross-linking to microtubules. In addition, the phosphorylation/dephosphorylation dependent regulation of NFH cross-linking to microtubules is disrupted following in vivo AlCl3 exposure by a mechanism that s independent of NFH/Al3+ binding.