Organophosphorus Compound–Induced Delayed Neurotoxicity in White Leghorn Hens Assessed by Fluoro-Jade

Abstract

Certain organophosphorus (OP) compounds can induce a delayed neuropathy, termed OPIDN, that involves central and peripheral nervous system axons, terminals, and perikarya. Historically, OPIDN has been characterized by staining neural sections with silver or hematoxylin and eosin (H&E). This study utilized a novel staining method, Fluoro-Jade, for evaluating the distribution and extent of OPIDN in the central nervous system of hens. Results were then compared to synoptically sectioned and stained H&E preparations. White Leghorn hens were injected with phenyl saligenin phosphate (PSP, 2.5 mg/kg, intramuscular [im]), triphenyl phosphite (TPPi, 500 mg/kg, subcutaneous [sc]), or dimethyl sulfoxide vehicle (DMSO, 0.5 ml/kg, im or sc) and evaluated clinically for signs of neurological dysfunction associated with OPIDN. Hens were sacrificed 7, 14, and 21 days post dosing. Brains and spinal cords were removed immediately following sacrifice, fixed in formalin, and embedded in paraffin. Microtomecut sections (7 μm) were then stained with Fluoro-Jade (0.001%, w/v) or H&E. Staining with Fluoro-Jade revealed time-dependent degeneration of nerve fibers and terminals (with PSP and TPPi), or cell bodies (with TPPi) in lamina VII, spinocerebellar, and medial pontine-spinal tracts of the lumbar spinal cord, in white matter and mossy fibers of foliae I–V and IX of the cerebellum, and in medullary, pontine, and midbrain nuclei and paleostriatal fibers surrounding the optic tract. TPPi-induced degeneration was more extensive than that induced by PSP and affected additional cerebellar folia, medullary, pontine, midbrain, and forebrain nuclei and fiber tracts. H&E-stained sections revealed fewer sites of neurodegeneration when compared to Fluoro-Jade. These results demonstrate that Fluoro-Jade is a sensitive method for staining neural tissue affected by OPIDN.

Exposure to certain organophosphorus (OP) compounds can initiate a progressive neuropathy, termed organophosphate-induced delayed neuropathy (OPIDN). This progressive neuropathy clinically affects a variety of species including adult hens, the U.S. Environmental Protection Agency (US EPA)-recommended animal model for studies involving acute and delayed neuropathy associated with OP compounds (US EPA 1998).

OPIDN is subdivided into two forms, type I and type II, on the basis of phosphate atom valence state and associated pathologic sequela (Abou-Donia 1995). OP compounds containing a pentavalant phosphorus, such as phenyl saligenin phosphate (PSP) and diisopropyl phosphorofluoridate (DFP), induce type I OPIDN. This form is characterized by distal axonal degeneration in peripheral nerves, long spinal tracts (such as the spinocerebellar tract), and some spinal, medullary, pontine, and cerebellar nuclei (Ehrich and Jortner 2001; Tanaka and Bursian 1989). OP compounds containing a trivalent phosphorus, such as triphenyl phosphite (TPPi), induce type II OPIDN. Type II OPIDN affects neuronal sites characterized for type I OPIDN and additional neuronal areas in the spinal cord, brainstem, midbrain, and fore-brain nuclei and tracts (Abou-Donia 1995; Ehrich and Jortner 2001; Tanaka, Bursian, and Lehning 1992; Varghese et al. 1995a, 1995b).

The distribution and extent of OPIDN have been assessed by both silver and hematoxylin and eosin (H&E) staining protocols in several model species, including hens (Tanaka and Bursian 1989; Tanaka, Bursian, and Lehning 1992), quail (Varghese et al. 1995a, 1995b), and ferrets (Stumpf et al. 1989; Tanaka et al. 1990; Tanaka, Bursian, and Aulerich 1994). Silver staining protocols selectively identify degenerating neuronal soma, axons (fibers), dendrites, and terminals through a largely unknown mechanism (Switzer 2000). H&E protocols result in blue-stained nuclei that are surrounded by cellular material that is primarily red, regardless of their tissue of origin. Silver staining is highly sensitive because of its specificity, but can have variable results (Switzer 2000; Schmued and Hopkins 2000). H&E, although more commonly used, is not specific for neurons and, therefore, requires adherence to specific criteria for making judgments in determining morphological changes in neurons. Ehrich and Jortner (2001) suggest that the perceived severity of OP-induced neurodegeneration is less when assessed with H&E staining than with silver protocols, even though the distribution of lesions is similar. H&E does have an advantage over silver staining, however, in that it permits the examination of cellular structure, a feature that is lost with silver staining.

Fluoro-Jade is a novel fluorescence stain that has been promoted for the detection of neuronal degeneration. It has been used to identify the distribution of neuronal cell death induced by exposure to xenobiotics (e.g., amphetamines, timethyl tin), and conditions such as sleep deprivation and intrastriatal nigral grafting procedures (Bowyer et al. 1998; Cirelli et al. 1999; Eisch, Schmued, and Marshall 1998; Emgard et al. 1999; Schmued et al. 1999; Schmued and Bowyer 1997; Schmued 2003). It is described as being more reliable, much simpler in technique, and comparatively as sensitive as silver staining procedures (Schmued, Albertson, and Slikker 1997). This staining method has also revealed additional neurodegeneration not identified with other methods (Freyaldenhoven, Ali, and Schmued 1997; Schmued and Hopkins 2000). The sensitivity, specificity, and ease of use suggest that Fluoro-Jade would be appropriate to use in the study of OPIDN.

This study compared and evaluated the temporal and spatial distribution of delayed neuropathy, as discerned by Fluoro-Jade and H&E, in the brain and spinal cord of white Leghorn hens following exposure to the OP compounds PSP and TPPi. Histopathological findings were correlated to clinical observations of neurological dysfunction.

METHODS

Chemicals

Triphenyl phosphite (TPPi; 96.7% pure, density 1.184 g/ml) was purchased from Chem Services (West Chester, PA). Phenyl saligenin phosphate (PSP; 99% pure) was synthesized by Lark Enterprises (Webster, MA). Dimethyl sulfoxide (DMSO) was obtained from Sigma (St. Louis, MO). Sodium pentobarbital was purchased from Veterinary Laboratories, Inc. (Lenexa, KS). OP compounds were diluted in DMSO the same day (TPPi, 1000 mg/ml) or diluted and stored at 23°C for 3 days (PSP, 5 mg/ml) prior to dosing hens.

Animal Maintenance, Dosing, Clinical Evaluation, and Sacrifice

White Leghorn hens (>10 months old, 1.5 to 1.9 kg) obtained from the Department of Animal and Poultry Science (Virginia Polytechnic Institute and State University) were housed in groups of four in wire cages (57 × 84 × 61 cm) and fed and watered ad libitum.

Nine hens were randomly assigned to each treatment group (PSP, 2.5 mg/kg; TPPi, 500 mg/kg; and DMSO, 0.5 ml/kg), dosed subcutaneously (DMSO, TPPi) or intramuscularly (DMSO, PSP) as in Tanaka, Bursian, and Lehning (1992) and Massicotte et al. (1999), and then returned to cages with similarly dosed birds. Hens within treatment groups were then assigned sacrifice dates randomly (7, 14, or 21 days). Hens disabled by paralysis at later time points were fed and watered manually on a daily basis.

Hens were observed daily for evidence of neurological dysfunction. Unblinded clinical evaluations were performed by one person and averaged for all three birds in the treatment/time set. Day 14 observations were verified by a person unfamiliar with the experimental setup. Observations were based on criteria modified from Stumpf et al. (1989). Numerical scores were designated: 0 = no deficits; 1 = slight leg weakness; 2 = leg weakness and reluctance to walk; 3 = mild ataxia; 4 = severe ataxia; 5 = mild paresis; and 6 = severe paresis.

On the sacrifice date, hens were euthanized with sodium pentobarbital (65 mg/ml) via a brachial wing vein. Immediately following, the brain with attached cervical spinal cord and the lumbar spinal cord were carefully dissected free, sagittally sectioned, and immersion fixed in ice-cold 10% neutral-buffered formalin.

Tissue Processing

Brain and lumbar spinal cord tissue were stored in formalin at 4°C for 4 days. Formalin-fixed samples (parasagittal sections of brain, coronal sections of lumbar spinal cord) were trimmed, dehydrated in a graded series of alcohols, cleared with xylene, and infiltrated with warm paraffin (56 to 63°C). Sections (7 μm thick) were cut from chilled paraffin blocks, attached to positively charged slides, and baked for 30 min at 60°C prior to staining.

Staining of Sections

Formalin-fixed sections were stained with H&E. Other formalin-fixed serial sections were stained with Fluoro-Jade, primarily as in Schmued and Hopkins (2000). Briefly, formalin-fixed sections were prepared for staining by deparaffinizing in xylene (15 min × 2), rehydrating in a graded series of ethanol, and rinsing in distilled water. Slides were then immersed for 15 min in freshly prepared 0.06% (w/v) potassium permanganate, rinsed in distilled water, and immersed in the dark for 30 min in a 0.001% (w/v) Fluoro-Jade staining solution (Histo-Chem Inc., Jefferson, AR). Stained slides were then rinsed in distilled water, dried by air convection in a chemical hood, dehydrated in xylene, and coverslipped with DPX (Aldrich Chemical Co; Milwaukee, WI). Blinded (H&E) and unblinded (Fluoro-Jade) observations of degenerating neurons were made. A Nikon Diaphot-TMD inverted microscope equipped with 10× and 20× fluorescence objectives and a “B” filter cube (~520 nm) was used. Photographic documentation of representative degenerative areas was made with a Nikon Fe-2 camera (ASA200 Kodacolor film). The location of brain and spinal cord tracts were identified using an atlas of the chick brain (Kuenzel and Masson 1988) and publications (Tanaka and Bursian 1989; Tanaka, Bursian, and Lehning 1992).

Data Analysis

Clinical observations had 3 to 9 data points for each time and OP compound. These data were graphed as the median ± the range. Qualitative histochemical studies had three samples for each time point. The findings from all samples within each treatment (n = 9) were combined into composite drawings to illustrate the entire amount of neuronal degeneration elicited by OP compounds over the entire duration of exposure (21 days).

RESULTS

Clinical Evaluation

No clinical deficits were observed in control hens following DMSO injections. A progression from leg weakness to ataxia and then to paralysis was observed over a period of 21 days in hens injected with PSP or TPPi. This was reflected by increases in clinical scores of neurological dysfunction from day 6 onward (Figure 1). Increased agitation and activity immediately following OP compound injections were also observed.

Histopathological Examination—Fluoro-Jade

Fluoro-Jade nonspecifically stained all brain tissue a dull yellow color when viewed under a fluorescence microscope. In contrast, degenerating neurons (fibers, terminals, soma) stained with substantially greater brightness and intensity. Degenerating neurons were morphologically distinct from other brightly stained non-neural entities (red blood cells, meninges, blood vessels, and choroid plexus) (Figure 2).

DMSO-injected hens displayed little or no increase in fluorescence intensity over background (Figures 3d , 4d ). In hens treated with PSP or TPPi, degenerating tracts (fibers), soma, and terminals stained much brighter than the surrounding dull fluorescent yellow matrix. The variation in stained regions within each treatment type and duration was greater in brain when compared to spinal cord sections.

The test agents induced a time-dependent degeneration of nerve fibers and terminals (PSP and TPPi), and cell bodies (TPPi) in lamina VII, spinocerebellar, and medial pontine-spinal tracts in the lumbar spinal cord (Figure 3e, f ). Staining of parasaggital brain sections from PSP- and TPPi-injected hens also revealed time-dependent heavy degeneration of cerebellar white matter and mossy fibers of foliae I–V and IX (Figure 4e, f ). Scattered degeneration was also observed in medullary, pontine, and midbrain nuclei and paleostriatal fibers surrounding the optic tract. TPPi alone induced additional degeneration in other cerebellar folia, medullary, pontine, midbrain, and forebrain nuclei and fiber tracts. Overall, fiber degeneration was more widespread in hens treated with TPPi. In brain structures other than the cerebellum and spinal cord, apparent degeneration peaked in density and distribution at day 14. Composite drawings of Fluoro-Jade stained neuronal degeneration observed 7, 14, or 21 days after administration of PSP and TPPi are presented in Figure 5 and Figure 6.

Histopathological Examination—H&E

The H&E method stained neuronal cell nuclei blue and cytological material pink. There was differential staining of cell bodies, neuropil, glial cells, and axons. No significant lesions were seen in controls (Figures 3a , 4a ). In the lumbar spinal cord of PSP-dosed hens, there were rare swollen axons in the region of the medial pontine spinal tract on day 14. By day 21, there were moderate numbers of such degenerating myelinated fibers in that region and in the spinocerebellar tracts (Figure 3b ). After 14 days, birds exposed to TPPi had degenerating myelinated fibers scattered through the lumbar cord white matter, manifest by swollen axons and fiber debris. This was especially prominent in the ventral white matter. The gray matter contained degenerating neuronal cell bodies, sometimes having a chromatolytic pattern. Neuronophagic foci (likely microglial) were seen around or replacing some of the necrotic neurons. Swollen neuronal processes, possibly axons, were also noted in the gray matter. These changes are prominent in the region of the lower motor neuron cell bodies in the ventral horn, but were also seen elsewhere in the gray matter. At day 21, degenerating fibers were seen in the spinocerebellar tracts and in longitudinally sectioned cord segments (Figure 3c ). Neuronal necrosis was noted as above, but neuronophagia was more prominent than on day 14.

PSP-dosed hens at 14 days demonstrated axonal swelling and myelinated degeneration in cervical cord and medullary white matter (mainly dorsal). These were also seen in the cerebellar subcortical and folial white matter. On day 21, similar but more advanced changes were noted (Figure 4b ). In TPPi-dosed hens on day 14, there was variable, often marked degeneration in multiple regions of the cervical spinal cord white matter, and myelinated tracts in the medulla and pons. These altered fibers sometimes extended into brainstem (especially pons) nuclei. The cerebellar subcortical and folial white matter had scattered fiber degeneration. In addition, occasional degeneration of neurons was seen in brainstem (especially pontine) nuclei. This was associated with microglial and lymphocytic infiltrates, which were prominent in one of three hens sacrificed at this time. On day 21 these lesions were more advanced (Figure 4c ).

In summary, both toxicants induced myelinated fiber degeneration. In PSP this was restricted to specific tracts where this could be determined (cross-sections of spinal cord), and no neuronal necrosis was seen. In TPPi-treated hens, the myelinated fiber degeneration was more widespread. In addition, there was associated necrosis of neurons, sometimes with associated neuronophagia. This was best developed in the lumbar spinal cord. The relative percentages of degeneration were 1% to 5% of fibers on H&E slides of OP-dosed hens and 10% to 25% and 25% to 50% on Fluoro-Jade slides of hens dosed with PSP and TPPi, respectively.

DISCUSSION

This study evaluated the onset and distribution of OPIDN in white Leghorn hens by utilizing Fluoro-Jade, a novel fluorochrome specific for localizing neuronal degeneration in axons, terminals and cell soma (Schmued and Hopkins 2000). Degenerative changes (increases in staining brightness) induced by PSP and TPPi were observed in brain and spinal cord tissue. Comparison was made with H&E staining in serial sections of the same thickness (7 μm). Fluoro-Jade was not used to stain sections of peripheral nerve, which are usually examined in 1-μm sections stained with toluidine blue and safranin (Ehrich and Jortner 2001; Jortner and Ehrich 1987; Jortner et al. 1989).

Overall, the Fluoro-Jade procedure appeared to discriminate OP compound–induced axonal and cell body injury in brain and spinal cord sections. Fluoro-Jade staining is not technically difficult, but does provide selectivity for injured neurons, although the details of cell structure are not demonstrated. It is recognized that, although it is a relatively simple procedure, H&E staining is not selective for neuronal degeneration.

In the present study, extensive PSP- and TPPi-induced neuronal degeneration was fluorescently labeled by Fluoro-Jade in brain and spinal cord sections. Degeneration in toto increased for both compounds up until day 14. Following this, a number of areas examined (except cerebellum and spinal cord) displayed reduced amount of degeneration (decreased staining of fibers, terminals, soma). Using silver staining, similar decreases in degeneration over time have been observed in medullary nuclei (vestibular, raphae magnus) of hens exposed to tri-ortho-tolyl phosphate (TOTP) (Tanaka and Bursian 1989), and auditory nuclei and tracts of quail exposed to TPPi (Varghese et al. 1995a). Decreases in degeneration with time have also been noted in toluidine blue-stained peripheral nerves of hens exposed to PSP or TOTP (Jortner et al. 1989). Apparent reductions in peripheral nervous system degeneration have been attributed to the clearance of neuronal tissue debris, and fiber regeneration.

For TPPi, which induces type II OPIDN, the distribution of degeneration was similar, but the extent of staining with Fluoro-Jade was less in lamina VII of the gray matter when compared to silver staining (Tanaka, Bursian, and Lehning 1992). In hens treated with TPPi, silver-stained degenerating areas were observed in the lateral reticular, lateral cervical, gracilecuneate, external cuneate, lateral paragigantocellular reticular, parvocellular, gigantocellular reticular, lateral vestibular, cerebellar deep, spiriform, and the lateral mesenphalic nucleus pars dorsalis (Tanaka, Bursian, and Lehning 1992). TPPi-induced neuronal degeneration, as determined by silver staining, has also been described in ferrets (Tanaka et al. 1990; Tanaka, Bursian, and Aulerich 1994) and quail (Varghese et al. 1995a, 1995b).

The distribution and density of PSP-induced spinal cord degeneration in spinocerebellar and medial-pontine tracts of hens as assessed by Fluoro-Jade was similar to that previously reported for type I OPIDN when using toluidine blue-safranin staining (Ehrich and Jortner 2001; Jortner and Ehrich 1987) or silver staining (Tanaka and Bursian 1989). Lesions in other areas of the central nervous system were more extensive with Fluoro-Jade and silver staining than previously described in hens using other staining methods. Findings could be based in interpretative or methodological differences, such as difficulties in precisely identifying the three-dimensional location of nuclei and fiber tracts. Thickness of stained specimens may also contribute to differences in staining distribution. For example, Tanaka and Bursian (1989) and Tanaka, Bursian, and Lehning (1992) utilized 40 μm thick sections for Fink-Heimer silver staining, in comparison to 7 μm thick sections used in this study. Observation of thicker sections would allow for higher confidence when designating structures, and should also theoretically reveal more information on neurodegenerative trends.

In this study, neural dysfunction was assessed by using a simplified method for scoring clinical observations. The manifestation of clinical dysfunction following PSP (2.5 mg/kg) exposure in this study was similar to that noted previously (El-Fawal et al. 1990; Jortner and Ehrich 1987). In addition, both onset and progression for symptoms induced by 500 mg/kg TPPi in the current study were similar to that of Tanaka et al. (1992) in which a 1000-mg/kg dosing strategy was used in hens. This suggested that a dose may exist beyond which TPPi may not induce any additional neurodegeneration in the hen. TPPi-induced clinical dysfunction in our hen model occurred much later and was less variable, however, than that reported by Varghese et al. (1995b) after the administration of 500 mg/kg to quail.

The results presented here are the first descriptions of the use of Fluoro-Jade as a discriminative staining technique for type I and type II OPIDN in hens. For this method, discrete staining, ease of use, and relatively low cost made Fluoro-Jade an excellent candidate for the assessment of neuropathic degeneration associated with exposure to OP compounds. It was more sensitive than H&E for identification of neuronal degeneration in the central nervous system. Results appeared consistent with, but less labor intensive, than silver staining (Switzer 2000; Schmued and Hopkins 2000).