Quantification of axonal loss in Alzheimer’s disease: an image analysis study

The density of axons in the optic nerve, olfactory tract and corpus callosum was quantified in non-demented elderly subjects and in Alzheimer’s disease (AD) using an image analysis system. In each fibre tract, there was significant reduction in the density of axons in AD compared with non-demented subjects, the greatest reductions being observed in the olfactory tract and corpus callosum. Axonal loss in the optic nerve and olfactory tract was mainly of axons with smaller myelinated cross-sectional areas. In the corpus callosum, a reduction in the number of ‘thin’ and ‘thick’ fibres was observed in AD, but there was a proportionally greater loss of the ‘thick’ fibres. The data suggest significant degeneration of white matter fibre tracts in AD involving the smaller axons in the two sensory nerves and both large and small axons in the corpus callosum. Loss of axons in AD could reflect an associated white matter disorder and/or be secondary to neuronal degeneration.

'Animal electricity' in the long eighteenth century with special reference to the Edinburgh medical school

Coleridge, looking back at the end of the ‘long eighteenth century’, remarked that the whole of natural philosophy had been ‘electrified’ by advances in the understanding of electrical phenomena. In this paper I trace the way in which these advances affected contemporary ‘neurophysiology.’ At the beginning of the long eighteenth century, neurophysiology (in spite of Swammerdam’s and Glisson’s demonstrations to the contrary) was still understood largely in terms of hollow nerves and animal spirits. At the end of that period the researches of microscopists and electricians had convinced most medical men that the old understanding had to be replaced. Walsh, Patterson, John Hunter and others had described the electric organs of electric fish. Gray and Nollet had demonstrated that electricity was not merely static, but flowed. Franklin had alerted the world to atmospheric electricity. Galvani’s frog experiments were widely known. Volta had invented his ‘pile.’ But did ‘animal electricity’ exist and was it identical to the electricity physicists studied in the inanimate world? Was the brain a gland, as Malpighi’s researches seemed to confirm., and did it secrete electricity into the nervous system? The Monros (primus and secundus), William Cullen, Luigi Galvani, Alessandro Volta, Erasmus Darwin, Luigi Rolando and François Baillarger all had their own ideas. This paper reviews these ‘long-eighteenth century’ controversies with special reference to the Edinburgh medical school and the interaction between neurophysiology and physics.

Large diameter optic nerve axon fibres in Alzheimer's disease

A variety of visual symptoms have been associated with Alzheimer's disease (AD). These include delays in flash visual evoked potentials which indicate a disruption of the integrity of the visual pathway. Examination of the visual cortex has revealed the presence of both senile plaques and neurofibrillary tangles. The purpose of this study was to determine whether there were differences in the number and/or size of optic nerve axons between AD patients and non-demented age-matched controls. Five optic nerves from AD patients and five from age-matched controls were embedded in epon resin and 1 micron sections prepared on a Reichert ultramicrotome. The sections were then stained in toluidine blue and examined at x400 magnification. The numbers of axons were counted in photographs of three fields taken at random from each section. To evaluate the axon diameters, 70 axons were chosen at random from each patient and measured using a calibrated eyepiece graticule. The total axon counts revealed no significant differences between the AD optic nerves and the age-matched controls. However, the frequency distribution of axon diameters was significantly different in the two groups. In particular, there were fewer larger diameter axons in patients with AD as previously reported. Degeneration of the large diameter axons suggests involvement of the magnocellular as opposed to the parvocellular pathways. Hence, there could be differences in visual performance of AD patients compared with normals which could be important in clinical diagnosis.

The incidence of corpora amylacea (CA) in the optic nerve of patients with Alzheimer's disease

Corpora amylacea (CA) are spherical or ovoid bodies 50-50 microns in diameter. They have been described in normal elderly brain as well as in a number of neurodegenerative disorders. In this study, the incidence of CA in the optic nerves of Alzheimer's disease (AD) patients was compared with normal elderly controls. Samples of optic nerves (MRC Brain Bank, Institute of Psychiatry) were taken from 12 AD patients (age range 69-94 years) and 18 controls (43-82 years). Optic nerves were fixed in 2% buffered glutaraldehyde, post-fixed in osmium tetroxide, embedded in epoxy resin and then sectioned to a thickness of 2 microns. Sections were stained with toluidine blue. CA were present in all of the optic nerves examined. In addition, a number of similarly stained but more irregularly shaped bodies were present. Fewer CA were found in the optic nerves of AD patients compared with controls. By contrast, the number or irregularly shaped bodies was increased in AD. In AD, there may be a preferential decline in the large diameter fibres which may mediate the M-cell pathway. Hence, the decline in the incidence of CA in AD may be associated with a reduction in these fibres. It is also possible that the irregualrly shaped bodies are a degeneration product of the CA.

Seeing and believing: three episodes in the history of neurophysiology

In this contribution I look at three episodes in the history of neurophysiology that bring out the complex relationship between seeing and believing. I start with Vesalius in the mid-sixteenth century who writes that he can in no way see any cavity in nerves, even in the optic nerves. He thus questions the age-old theory (dating back to the Alexandrians in the third century BC) but, because of the overarching psychophysiology of his time, does not press his case. This conflict between observation and theory persisted for a quarter of a millennium until finally resolved at the beginning of the nineteenth century by the discoveries of Galvani and Volta. The second case is provided by the early history of retinal synaptology. Schultze in 1866 had represented rod spherules and bipolar dendrites in the outer plexiform layer as being separated by a (synaptic) gap, yet in his written account, because of his theoretical commitments, held them to be continuous. Cajal later, 1892, criticized Schultze for this pusillanimity, but his own figure in La Cellule is by no means clear. It was only with the advent of the electron microscopy in the mid-twentieth century that the true complexity of the junction was revealed and it was shown that both investigators were partially right. My final example comes from the Hodgkin-Huxley biophysics of the 1950s. Their theory of the action potential depended on the existence of unseen ion pores with quite complex biophysical characteristics. These were not seen until the Nobel-Prize-winning X-ray diffraction analyses of the early twenty-first century. Seeing, even at several removes, then confirmed Hodgkin and Huxley’s belief. The relation between seeing and believing is by no means straightforward.

The neurotoxicity of acrylamide

The experiments described in this thesis compared conventional methods of screening for neurotoxins with potential electrophysiological and pharmacological tests in an attempt to improve the sensitivity of detection of progressive distal neuropathy. Adult male albino mice were dosed orally with the neurotoxicant acylamide and subjected to a test of limb strength and co-ordination and a functional observational battery. These methods established a no observable effect level of 10 mg/kg. A dose of 200 mg/kg resulted in abnormalities of gait and reduced limb strength and/or co-ordination. Analysis of the in vitro 'jitter' of the latency of trains of action potentials evoked at a frequency of 30 Hz in the mouse phrenic nerve/hemidiaphragm preparation showed this technique to be unsuitable for detection of the early phases of acrylamide induced peripheral neuropathy (l00 mg/kg). The evoked and spontaneous twitch responses of the hemidiaphragm preparation following in vitro exposure to the organophosphorous anticholinesterase compound ecothiopate were altered by in vivo pre treatment with acrylamide. Acrylamide caused an increase in the time course of the potentiation of stimulated twitches and a decrease in the maximum potentiation. Spontaneous twitches were reduced in amplitude and frequency. These effects occurred at an acrylamide dose level insufficient to cause clinical signs of neuropathy. Investigations into the mechanisms underlying these observations yielded the following observations. Analysis of miniature endplate potentials at this dose level indicated prolongation of the life of acetylcholine in the synaptic cleft but the implied decrease in cholinesterase activity could not be demonstrated biochemically or histologically. The electrical excitability of the nerve terminal region of phrenic motor nerves was reduced following acrylamide although a possible compromise of antidromic action potential conduction could not be confirmed. There was no histopathological evidence of neuropathy at this dose level. Further exploration of this phenomenon is desirable in order to ascertain whether the effect is specific to acrylamide and/or ecothiopate and to elucidate the mechanisms behind these novel observations.

A quantitative analysis of optic nerve axons in elderly control subjects and patients with Alzheimer's disease

Objective: To study the density and cross-sectional area of axons in the optic nerve in elderly control subjects and in cases of Alzheimer's disease (AD) using an image analysis system. Methods: Sections of optic nerves from control and AD patients were stained with toluidine blue to reveal axon profiles. Results: The density of axons was reduced in both the center and peripheral portions of the optic nerve in AD compared with control patients. Analysis of axons with different cross-sectional areas suggested a specific loss of the smaller sized axons in AD, i.e., those with areas less that 1.99 μm2. An analysis of axons >11 μm2 in cross-sectional area suggested no specific loss of the larger axons in this group of patients. Conclusions: The data suggest that image analysis provides an accurate and reproducible method of quantifying axons in the optic nerve. In addition, the data suggest that axons are lost throughout the optic nerve with a specific loss of the smaller-sized axons. Loss of the smaller axons may explain the deficits in color vision observed in a significant proportion of patients with AD.

The presence of pleiotrophin in the human intervertebral disc is associated with increased vascularization:an immunohistologic study

Study Design. An immunohistological study of surgical specimens of human intervertebral disc.Objective.To examine the presence of pleiotrophin in diseased or damaged intervertebral disc tissue and the association between its presence and the extent of tissue vascularization and innervation.Summary of Background Data. Increased levels of pleiotrophin, a growth and differentiation factor that is active in various pathophysiologic processes, including angiogenesis, has been associated with osteoarthritic changes of human articular cartilage. The association between pleiotrophin expression and pathologic conditions of the human intervertebral disc is unknown.Methods. Specimens of human lumbar intervertebral discs, obtained following surgical discectomy, were divided into 3 groups: nondegenerated discs (n = 7), degenerated discs (n = 6), and prolapsed discs (n = 11). Serial tissue sections of each specimen were immunostained to determine the presence of pleiotrophin, blood vessels (CD34-positive endothelial cells), and nerves (neurofilament 200 kDa [NF200]-positive nerve fibers).Results. Pleiotrophin immunoreactivity was seen in disc cells, endothelial cells, and in the extracellular matrix in most specimens of intervertebral disc but was most prevalent in vascularized tissue in prolapsed discs. There was a significant correlation between the presence of pleiotrophin-positive disc cells and that of CD34-positive blood vessels. NF200-positive nerves were seen in vascularized areas of more degenerated discs, but nerves did not appear to codistribute with blood vessels or pleiotrophin positivity in prolapsed discs.Conclusions. Pleiotrophin is present in pathologic human intervertebral discs, and its prevalence and distribution suggest that it may play a role in neovascularization of diseased or damaged disc tissue.

Human intervertebral disc cells promote nerve growth over substrata of human intervertebral disc aggrecan

Study Design. Coculture assays of the migration and interaction of human intervertebral disc cells and chick sensory nerves on alternate substrata of collagen and aggrecan. Objective. To examine the effects of aggrecan on disc cell migration, how disc cells and sensory nerves interact, and whether disc cells affect previously reported inhibitory effects of aggrecan on sensory nerve growth. Summary of Background Data. Human intervertebral disc aggrecan is inhibitory to sensory nerve growth in vitro, suggesting that a loss of aggrecan from the disc may have a role in the increased innervation seen in disc degeneration. Endothelial cells that appear to co-migrate with nerves into degenerated intervertebral disc express neurotrophic factors, but the effects of disc cells on nerve growth are not known. Methods. Human disc cells were seeded onto tissue culture plates that had been coated with type I collagen and human intervertebral disc aggrecan. Explants of chick dorsal root ganglions (DRGs) were subsequently added to the plates and sensory neurite outgrowth stimulated by the addition of nerve growth factor. Time-lapse video and fluorescence microscopy were used to examine the migration and interaction of the disc cells and sensory neurites, in the context of the different matrix substrata. The effects of disc cell conditioned medium on nerve growth were also examined. Results. Disc cells spread and migrated on collagen until they encountered the aggrecan substrata, where some cells, but not all, were repelled. In coculture, DRG neurites extended onto the collagen/disc cells until they encountered the aggrecan, where, like the disc cells, many were repelled. However, in the presence of disc cells, some neurites were able to cross onto this normally inhibitory substratum. The number of neurite crossings onto aggrecan correlated significantly with the number of disc cells present on the aggrecan. In control experiments using DRG alone, all extending neurites were repelled at the collagen/aggrecan border. Conditioned medium from disc cell cultures stimulated DRG neurite outgrowth on collagen but did not increase neurite crossing onto aggrecan substrata. Conclusions. Human disc cells migrate across aggrecan substrata that are repellent to sensory DRG neurites. Disc cells synthesize neurotrophic factors in vitro that promote neurite outgrowth. Furthermore, the presence of disc cells in coculture with DRG partially abrogates the inhibitory effects of aggrecan on nerve growth. These findings have important implications for the regulation of nerve growth into the intervertebral disc, but whether disc cells promote nerve growth in vivo remains to be determined.

Human intervertebral disc aggrecan inhibits endothelial cell adhesion and cell migration in vitro

STUDY DESIGN: The effect of human intervertebral disc aggrecan on endothelial cell growth was examined using cell culture assays. OBJECTIVE: To determine the response of endothelial cells to human intervertebral disc aggrecan, and whether the amount and type of aggrecan present in the intervertebral disc may be implicated in disc vascularization. SUMMARY OF BACKGROUND DATA: Intervertebral disc degeneration has been associated with a loss of proteoglycan, and the ingrowth of blood vessels and nerves. Neovascularization is a common feature also of disc herniation. Intervertebral disc aggrecan is inhibitory to sensory nerve growth, but the effects of disc aggrecan on endothelial cell growth are not known. METHODS: Aggrecan monomers were isolated separately from the anulus fibrosus and nucleus pulposus of human lumbar intervertebral discs, and characterized to determine the amount and type of sulfated glycosaminoglycan side chains present. The effects of these aggrecan isolates on the cellular adhesion and migration of the human endothelial cell lines, HMEC-1 and EAhy-926, were examined in vitro. RESULTS: Homogenous substrata of disc aggrecan inhibited endothelial cell adhesion and cell spreading in a concentration dependent manner. In substrata choice assays, endothelial cells seeded onto collagen type I migrated over the collagen until they encountered substrata of disc aggrecan, where they either stopped migrating, retreated onto the collagen, or, more commonly, changed direction to align along the collagen-aggrecan border. The inhibitory effect of aggrecan on endothelial cell migration was concentration dependent, and reduced by enzymatic treatment of the aggrecan monomers with a combination of chondroitinase ABC and keratinase/keratinase II. Anulus fibrosus aggrecan was more inhibitory to endothelial cell adhesion than nucleus pulposus aggrecan. However, this difference did not relate to the extent to which the different aggrecan isolates were charged, as determined by colorimetric assay with 1,9-dimethylmethylene blue, or to marked differences in the distribution of chondroitin sulfated and keratan sulfated side chains. CONCLUSIONS: Human intervertebral disc aggrecan is inhibitory to endothelial cell migration, and this inhibitory effect appears to depend, in part, on the presence of glycosaminoglycan side chains on the aggrecan monomer.

Diabetes and contact lens wear

The literature suggests that diabetic patients may have altered tear chemistry and tear secretion as well as structural and functional changes to the corneal epithelium, endothelium and nerves. These factors, together with a reported increased incidence of corneal infection, suggest that diabetic patients may be particularly susceptible to developing ocular complications during contact lens wear. Reports of contact lens-induced complications in diabetic patients do exist, although a number of these reports concern patients with advanced diabetic eye disease using lenses on an extended wear basis. Over the past decade or so, there have been published studies documenting the response of the diabetic eye to more modern contact lens modalities. The results of these studies suggest that contact lenses can be a viable mode of refractive correction for diabetic patients. Furthermore, new research suggests that the measurement of tear glucose concentration could, in future, be used to monitor metabolic control non-invasively in diabetic patients. This could be carried out using contact lenses manufactured from hydrogel polymers embedded with glucose-sensing agents or nanoscale digital electronic technology. The purpose of this paper is to review the literature on the anterior ocular manifestations of diabetes, particularly that pertaining to contact lens wear. © 2012 The Authors. Clinical and Experimental Optometry © 2012 Optometrists Association Australia.

Pupillary shapes in response to electrical stimulation of the sclera of peripheral cornea in cats and porcines

Purpose: We have reported that the changes in the pupillary shape in response to electrical stimulation of the branches of the ciliary nerves in cats. (Miyagawa et al. PLoS One, 2014). This study investigates the changes in the pupillary shapes in response to electrical stimulations of the sclera of peripheral cornea in cats and porcines. Methods: Two enucleated eyes of two cats and three enucleated porcine eyes were studied. Trains of biphasic pulses (current, 3 mA; duration, 2 ms/phase; frequency, 40 Hz) were applied using a tungsten electrode (0.3mm diameter). The stimulation was performed at every 45 degree over the entire circular region on the sclera near the cornea. The pupillary images were recorded before and 4 s (cat) and 10 s (pig) after the stimulation and the change in the pupil diameter (Δr) was quantified. The pupillary images were obtained with a custom-built compact wavefront aberrometer (Uday et al. J Cataract Refract Surg, 2013). Results: In a cat eye, the pupil was dilated by the electrical stimulation at six out of eight orientations (before stimulation pupil diameter r=10.10±0.49 mm, Δr=0.33±0.12 mm). The pupil dilated only toward the electrode (relative eccentricity of the pupil center to the pupil diameter change amount rdec=1.15±0.28). In the porcine eyes, the pupils were constricted by the electrical stimulations at the temporal and nasal orientations (r=10.04±0.57 mm, Δr=1.52±0.70 mm). The pupils contracted symmetrically (rdec=0.30±0.12). Conclusions: With electrical stimulation in the sclera of the peripheral cornea, asymmetric mydriasis in cat eyes and symmetrical miosis in porcine eyes were observed. Under the assumption that the electrical stimulation stimulated both muscles that contribute to the pupil control, our hypothesis proposed here is that the pupil dilator is stronger than the pupil sphincter in cat, and pupil sphincter is stronger than pupil dilator in porcine.

Accommodative response to electrical stimulation of the sclera of peripheral cornea in cats and porcines

Purpose: We have reported that the changes in the accommodative response to electrical stimulation of the branches of the ciliary nerves in cats. (Miyagawa et al, PLoS One, 2014). We have also reported that no robust accommodative responses to the electrical stimulations of the sclera of peripheral cornea (SSPC) were observed in enucleated porcine eyes (Mihashi et al, VPOptics, 2014). In this study, accommodative responses to SSPC stimulation in cats and porcines were investigated. Methods: Two eyes of two cats under anesthesia and after they were sacrificed were studied. Three enucleated porcine eyes obtained from a local slaughterhouse were also studied. Trains of biphasic pulses (current, 3 mA; duration, 2 ms/phase; frequency, 40 Hz) were applied using a tungsten electrode (0.3mm diameter) from several orientations. Wavefront sensing with a compact wavefront aberrometer (Uday et al J Cataract Refract Surg, 2013) were performed before and 4 s (cat) and 10 s (pig) after the stimulations and wavefront aberrations including spherical errors were analyzed over a 4-mm pupil area. Results: In the first cat under anesthesia, at three out of seven stimulus positions, 0.2 D hyperopic accommodative responses were observed and in two orientations, myopic responses were observed. For the other cat, weak accommodative responses including astigmatic changes were observed. In the sacrificed condition of the second cat, 0.1 D myopic response was observed for one stimulus orientation and the smaller responses were observed at six out of eight stimulus positions. No accommodative responses were elicited for the enucleated porcine eyes. Conclusions: In the anesthetized cats, electrical stimulation of the SSPC induced accommodative responses; the responses were unstable and weaker than the responses by the ciliary nerve stimulations we observed in our previous study. Small accommodative responses were observed after one of two cats had been sacrificed, but no accommodative responses were detected in the enucleated porcine eyes. Further studies are needed to confirm difference in the accommodation functions in the two species.