DSP-01 Barriers to the diagnosis of motor neuron disease - a South Australian study.

Background: MND is a progressive neurodegenerative disease characterised by death of upper and lower motor neurons, leading to progressive weakness of the bulbar, limb, thoracic and abdominal muscles. MND has a fairly stereotypical course, with death from respiratory failure occurring 2-4 years after symptom onset in most cases (1). Making the diagnosis of MND can be straightforward when key clinical criteria are met; however, at first presentation, rarely do patients meet these criteria, neurological changes may be subtle and disease progression slow. Thus, diagnosis poses a significant challenge, particularly in general practice, where patients are most likely to first present. Not surprisingly, there is usually a delay of 10-18 months between symptom onset and MND diagnosis (2). Importantly, early assessment by a neurologist is associated with a shorter time to MND diagnosis (3), which has significant implications for access to healthcare, including Riluzole and multidisciplinary clinics, which improve survival (4). Although delay in diagnosis is well documented, there have been no studies that have sought to identify factors associated with time to diagnosis, thereby enabling targeted implementation of a public health intervention.Objectives: To characterise the clinical factors that influence time to diagnosis of MND.Methods: 112 patients with MND attending the Southern Adelaide MND Clinic enrolled between January 2016 - 2018 were retrospectively recruited in to a cohort study. Information pertaining to the patient's demographics and their journey to diagnosis collected by a specialist physician and stored in the Australian MND Registry during clinic review were analysed to identify factors associated with time to diagnosis.Results: Mean time to diagnosis was 13 ± 1 months (range 1 - 38 months) from symptom onset. 41% of patients were classified as having fast disease progression; compared to those with slow disease progression, these patients were diagnosed earlier (8 ± 1 months vs 16 ± 2 months) (p < 0.0001, t = 34.6, df =220), were less likely to undergo multiple specialist opinions prior to referral to a neurologist (53% vs 73%) (p < 0.05, Chi-squared = 9.5, df =1), and were more disabled at time of diagnosis (mean ALSFRS-R 33 ± 5 vs ALSFRS-R 41 ± 5) (p < 0.0001, t = 12.4, df =220).Discussion and conclusions: Fast disease progression identifies a dichotomy of MND patients diagnosed earlier, although more disabled at diagnosis, likely mediated by a more efficient referral process. A greater awareness of MND is required to shorten time to diagnosis.

Factors contributing to delays in the diagnosis of motor neuron disease - A South Australian study.

OBJECTIVE(S): To characterize the clinical factors that influence time to diagnosis of motor neuron disease (MND) in a cohort of patients living in South Australia. DESIGN: A retrospective study. SETTING: Single centre study of patients managed at a tertiary referral hospital. PARTICIPANTS: Patients with MND living in South Australia enrolled in the Australian MND Registry between January 2016 and January 2018. One participant was excluded as study variables of interest were missing. RESULTS: The mean time to diagnosis was 13 ±â€¯1 months (median 11 months; range 1-38 months) from symptom onset. 41% of patients were classified as having fast disease progression; mean age of disease onset of those with fast disease progression was significantly later in life compared to those with slow disease progression (68 ±â€¯10 years vs 64 ±â€¯8 years) (P < .05, t = -3.921, df = 220). Patients with fast disease progression were diagnosed significantly earlier than those with slow disease progression (8 ±â€¯1 months vs 16 ±â€¯2 months) (P < .0001, t = 34.6, df = 220), were less likely to undergo multiple specialist opinions prior to referral to a neurologist (53% vs 73%) (P < .05, Chi-squared = 9.5, df = 1), and were significantly more disabled at time of diagnosis (mean ALSFRS-R 33 ±â€¯5) than those with slow disease progression (mean ALSFRS-R 41 ±â€¯5) (P < .0001, t = 12.4, df = 220). CONCLUSION(S): Fast disease progression identifies a dichotomy of MND patients that are diagnosed earlier, probable because they are more disabled at diagnosis, likely mediated by a more efficient referral process. A greater awareness of the disease and increased accessibility to neurologists is required to shorten time to diagnosis.

Neurochemical characterization of extrinsic nerves in myenteric ganglia of the guinea pig distal colon.

Extrinsic nerves to the gut influence the absorption of water and electrolytes and expulsion of waste contents, largely via regulation of enteric neural circuits; they also contribute to control of blood flow. The distal colon is innervated by extrinsic sympathetic and parasympathetic efferent and spinal afferent neurons, via axons in colonic nerve trunks. In the present study, biotinamide tracing of colonic nerves was combined with immunohistochemical labeling for markers of sympathetic, parasympathetic, and spinal afferent neurons to quantify their relative contribution to the extrinsic innervation. Calcitonin gene-related peptide, vesicular acetylcholine transporter, and tyrosine hydroxylase, which selectively label spinal afferent, parasympathetic, and sympathetic axons, respectively, were detected immunohistochemically in 1 ± 0.5% (n = 7), 15 ± 4.7% (n = 6), and 24 ± 4% (n = 7) of biotinamide-labeled extrinsic axons in myenteric ganglia. Immunoreactivity for vasoactive intestinal polypeptide, nitric oxide synthase, somatostatin, and vesicular glutamate transporters 1 and 2 accounted for a combined maximum of 14% of biotinamide-labeled axons in myenteric ganglia. Thus, a maximum of 53% of biotinamide-labeled extrinsic axons in myenteric ganglia were labeled by antisera to one of these eight markers. Viscerofugal neurons were also labeled by biotinamide. They had distinct morphologies and spatial distributions that correlated closely with their immunoreactivity for nitric oxide synthase and choline acetyltransferase. As reported for the rectum, nearly half of all extrinsic nerve fibers to the distal colon lack the key immunohistochemical markers commonly used for their identification. Their abundance may therefore have been significantly underestimated in previous immunohistochemical studies.

Selective coexpression of synaptic proteins, α-synuclein, cysteine string protein-α, synaptophysin, synaptotagmin-1, and synaptobrevin-2 in vesicular acetylcholine transporter-immunoreactive axons in the guinea pig ileum.

Parkinson's disease is a neurodegenerative disorder characterized by Lewy bodies and neurites composed mainly of the presynaptic protein α-synuclein. Frequently, Lewy bodies and neurites are identified in the gut of Parkinson's disease patients and may underlie associated gastrointestinal dysfunctions. We recently reported selective expression of α-synuclein in the axons of cholinergic neurons in the guinea pig and human distal gut; however, it is not clear whether α-synuclein expression varies along the gut, nor how closely expression is associated with other synaptic proteins. We used multiple-labeling immunohistochemistry to quantify which neurons in the guinea pig ileum expressed α-synuclein, cysteine string protein-α (CSPα), synaptophysin, synaptotagmin-1, or synaptobrevin-2 in their axons. Among the 10 neurochemically defined axonal populations, a significantly greater proportion of vesicular acetylcholine transporter-immunoreactive (VAChT-IR) varicosities (80% ± 1.7%, n = 4, P < 0.001) contained α-synuclein immunoreactivity, and a significantly greater proportion of α-synuclein-IR axons also contained VAChT immunoreactivity (78% ± 1.3%, n = 4) compared with any of the other nine populations (P < 0.001). Among synaptophysin-, synaptotagmin-1-, synaptobrevin-2-, and CSPα-IR varicosities, 98% ± 0.7%, 96% ± 0.7%, 88% ± 1.6%, and 85% ± 2.9% (n = 4) contained α-synuclein immunoreactivity, respectively. Among α-synuclein-IR varicosities, 96% ± 0.9%, 99% ± 0.6%, 83% ± 1.9%, and 87% ± 2.3% (n = 4) contained synaptophysin-, synaptotagmin-1-, synaptobrevin-2-, and CSPα immunoreactivity, respectively. We report a close association between the expression of α-synuclein and the expression of other synaptic proteins in cholinergic axons in the guinea pig ileum. Selective expression of α-synuclein may relate to the neurotransmitter system utilized and predispose cholinergic enteric neurons to degeneration in Parkinson's disease.

Selective expression of α-synuclein-immunoreactivity in vesicular acetylcholine transporter-immunoreactive axons in the guinea pig rectum and human colon.

Parkinson's disease is a neurodegenerative disorder characterized by motor and nonmotor impairments, including constipation. The hallmark pathological features of Parkinson's disease are Lewy bodies and neurites, of which aggregated α-synuclein is a major constituent. Frequently, Lewy pathology is identified in the distal gut of constipated Parkinson's disease patients. The neurons that innervate the distal gut that express α-synuclein have not been identified. We used multiple-labeling immunohistochemistry and anterograde tracing to quantify which neurons projecting to the guinea pig rectum and human colon expressed α-synuclein in their axons. α-Synuclein-immunoreactivity was present in 24 ± 0.7% of somatostatin (SOM)-immunoreactive (IR) varicosities; 20 ± 4.3% of substance P (SP)-IR varicosities and 9 ± 1.3% vasoactive intestinal polypeptide (VIP)-IR varicosities in guinea pig rectal myenteric ganglia. However, α-synuclein-immunoreactivity was localized in significantly more vesicular acetylcholine transporter (VAChT)-IR varicosities (88 ± 3%, P < 0.001). Of SOM-IR, SP-IR, and VIP-IR varicosities that lacked VAChT-immunoreactivity, only 1 ± 0.3%, 0 ± 0.3%, and 0% contained α-synuclein-immunoreactivity, respectively. 71 ± 0.8% of VAChT-IR varicosities in myenteric ganglia of human colon were α-synuclein-IR. In guinea pig rectal myenteric ganglia, α-synuclein- and VAChT-immunoreactivity coexisted in 15 ± 1.4% of biotinamide-labeled extrinsic varicosities; only 1 ± 0.3% of biotinamide-labeled extrinsic varicosities contained α-synuclein-immunoreactivity without VAChT-immunoreactivity. α-Synuclein expression in axons to the distal gut correlates closely with expression of the cholinergic marker, VAChT. This is the first report of cell-selective α-synuclein expression in the nervous system. Our results suggest cholinergic neurons in the gut may be vulnerable in Parkinson's disease.

Neurochemical coding compared between varicose axons and cell bodies of myenteric neurons in the guinea-pig ileum.

The discrete functional classes of enteric neurons in the mammalian gastrointestinal tract have been successfully distinguished on the basis of the unique combination of molecules and enzymes in their cell bodies ("chemical coding"). Whether the same chemical coding exists in varicose axons of different functional classes has not been systematically tested. In this study, we quantified the coexistence of markers that define classes of nerve cell bodies in the myenteric plexus of the guinea-pig ileum, in varicose axons of the same neurons. Profound differences between the combinations of immunohistochemical markers in myenteric nerve cell bodies and in their varicosities were identified. These discrepancies were particularly notable for classes of neurons that had previously been classified as cholinergic, based on immunoreactivity for choline acetyltransferase (ChAT) in their cell bodies. To detect cholinergic varicose axons of enteric neurons in this study, we used antiserum against the vesicular acetylcholine transporter (VAChT). ChAT-immunoreactivity has been reported to be consistently co-localized with 5-hydroxytryptamine (5-HT) in interneuronal cell bodies, yet only 29±5% (n=4) of 5-HT-immunoreactive varicosities contained vesicular acetylcholine transporter (VAChT). Somatostatin coexists with ChAT-immunoreactivity in a class of descending interneuron but only 21±1% (n=4) of somatostatin-immunoreactive varicosities were VAChT-immunoreactive. Comparable discrepancies were also noted for non-cholinergic markers. The results suggest that chemical coding of cell bodies does not necessarily reflect chemical coding of varicose axon terminals and that the assumption that nerve cell bodies that contain ChAT are functionally cholinergic may be questionable.