Karl F. Jensen

Chronic administration of aluminum–fluoride or sodium–fluoride to rats in drinking water: alterations in neuronal and cerebrovascular integrity

This study describes alterations in the nervous system resulting from chronic administration of the fluoroaluminum complex (AlF3) or equivalent levels of fluoride (F) in the form of sodium-fluoride (NaF). Twenty seven adult male Long-Evans rats were administered one of three treatments for 52 weeks: the control group was administered double distilled deionized drinking water (ddw). The aluminum-treated group received ddw with 0.5 ppm AlF3 and the NaF group received ddw with 2.1 ppm NaF containing the equivalent amount of F as in the AlF3 ddw. Tissue aluminum (Al) levels of brain, liver and kidney were assessed with the Direct Current Plasma (DCP) technique and its distribution assessed with Morin histochemistry. Histological sections of brain were stained with hematoxylin & eosin (H&E), Cresyl violet, Bielschowsky silver stain, or immunohistochemically for beta-amyloid, amyloid A, and IgM. No differences were found between the body weights of rats in the different treatment groups although more rats died in the AlF3 group than in the control group. The Al levels in samples of brain and kidney were higher in both the AlF3 and NaF groups relative to controls. The effects of the two treatments on cerebrovascular and neuronal integrity were qualitatively and quantitatively different. These alterations were greater in animals in the AlF3 group than in the NaF group and greater in the NaF group than in controls.

STP Position Paper Recommended Practices for Sampling and Processing the Nervous System (Brain, Spinal Cord, Nerve, and Eye) during Nonclinical General Toxicity Studies

The Society of Toxicologic Pathology charged a Nervous System Sampling Working Group with devising recommended practices to routinely screen the central nervous system (CNS) and peripheral nervous system (PNS) in Good Laboratory Practice–type nonclinical general toxicity studies. Brains should be weighed and trimmed similarly for all animals in a study. Certain structures should be sampled regularly: caudate/putamen, cerebellum, cerebral cortex, choroid plexus, eye (with optic nerve), hippocampus, hypothalamus, medulla oblongata, midbrain, nerve, olfactory bulb (rodents only), pons, spinal cord, and thalamus. Brain regions may be sampled bilaterally in rodents using 6 to 7 coronal sections, and unilaterally in nonrodents with 6 to 7 coronal hemisections. Spinal cord and nerves should be examined in transverse and longitudinal (or oblique) orientations. Most Working Group members considered immersion fixation in formalin (for CNS or PNS) or a solution containing acetic acid (for eye), paraffin embedding, and initial evaluation limited to hematoxylin and eosin (H&E)-stained sections to be acceptable for routine microscopic evaluation during general toxicity studies; other neurohistological methods may be undertaken if needed to better characterize H&E findings. Initial microscopic analyses should be qualitative and done with foreknowledge of treatments and doses (i.e., “unblinded”). The pathology report should clearly communicate structures that were assessed and methodological details. Since neuropathologic assessment is only one aspect of general toxicity studies, institutions should retain flexibility in customizing their sampling, processing, analytical, and reporting procedures as long as major neural targets are evaluated systematically.

Best Practices for Reporting Pathology Interpretations within GLP Toxicology Studies

Pfizer, Inc., Groton, Connecticut 06340, USA Merck Research Laboratories, West Point, Pennsylvania 19586 USA Pathology Branch, Center for Food Safety and Applied Nutrition, FDA, College Park, Maryland 20740, USA U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA Charles River Laboratories, Senneville, Quebec H9X 3R3, Canada Merck and Company, Inc., West Point, Pennsylvania 19486, USA Wyeth Research, Chazy, New York 12921 USA Health Canada Ottawa, Ontario K1A 0L2, Canada Sanofi-Aventis, Porcheville 78440, France Vet Path Services, Inc., Cincinnati, Ohio 45249, USA Albert Einstein College of Medicine Bronx, New York 10461, USA Purdue University, West Lafeyette, Indiana 47907, USA

Focal lesions of visual cortex—Effects on visual evoked potentials in rats

Focal lesions were placed in the visual cortex of Long-Evans hooded rats, immediately below skull screw recording electrodes. Lesions were produced by heat, and extended an average depth of about 0.9 mm below the cortical surface. Evoked potentials recorded from the electrode overlying the cortical lesion were compared with simultaneously recorded potentials from a contralateral homotopic site. The effects of the lesion were selective. Flash-evoked potential peaks P1, P2, and N2 were depressed by the lesion, and peaks N1 and P3 were augmented; peak N3 was unaffected. Pattern reversal evoked potential peak N3 was depressed by the lesion, and peaks N1 and P2 were made more distinct. The results emphasized that different peaks have different generators, and suggest in particular that flash-evoked potential peaks P1 and N2, and peak N3 of the pattern reversal-evoked potential require the superficial layers of the cortex.

Toxin‐Induced Blood Vessel Inclusions Caused by the Chronic Administration of Aluminum and Sodium Fluoride and Their Implications for Dementiaa

Until our knowledge of the etiology of Alzheimers dementia, as well as related conditions involving mental impairments, is greatly extended, no line of investigation should be ignored. We believed that the possible contributions of aluminum exposure to neural impairments deserved further study. In coming to this opinion we were mindful of the work of Roberts on the neurotoxic effects of inhaled aluminum silicate as well as the neuropathologic results reported by Per1 and his associates indicating an association of aluminum with disease-affected neurons in Alzheimers patients.*. The possibility that certain metals including aluminum, either alone or in combination, play a role in dementia remains a viable hypothesisP While the possibility of transport of A1 to the brain via the olfactory system, especially under conditions of a partially compromised immune system, remains a likely route of entry into the nervous system, it is not the only entry route for aluminum. A1 and other elements with toxic potential enter the nervous system through many pathways, including our food and water. Initially, we investigated the effects of low doses of aluminum given to rats through their drinking water. The entry of A1 into the circulation and the brain depends on the particular species of A1 available as well as the conditions in the stomach and the digestive tract. The bioavailability of A1 may be enhanced by its complexing with fluorine (F) to form various monomeric fluoaluminum species. Of these, AIF3 was of special interest due to its lipid solubility and ability to pass

Neuroanatomical Techniques for Labeling Neurons and Their Utility in Neurotoxicology

Publisher Summary This chapter describes neuroanatomical methods suited for labeling neurons. The chapter also addresses the importance of neuroanatomical approaches in ascertaining the significance of structural alterations. The chapter demonstrates how neuro-anatomical approaches, when applied in conjunction with basic toxicological considerations, can reveal how toxicants alter the structure of the nervous system. Morphological approaches are unique in their capacity to provide detailed information about the location and extent of toxicant-induced damage. This kind of information is critical for recognizing the significance of structural alterations in pathogenesis. By interpreting toxicant-induced alterations in terms of their impact on neural circuitry, an important link can be established between neuropathology and functional deficits. This chapter discusses the assessment of neural circuitry at three levels of organization. The first level corresponds to major brain regions and their interconnecting pathways. The second level is the individual neuron. The third level is the subcellular organization of molecules critical to neuronal integrity. For the methods applied to each of these three levels, this chapter briefly describes the methods, their advantages and disadvantages with regard to making inferences concerning neural structure and provides examples where such methods have been applied to the assessment of neurotoxicity or related injury.

Generation and characterization of neurogenin1-GFP transgenic medaka with potential for rapid developmental neurotoxicity screening.

Fish models such as zebrafish and medaka are increasingly used as alternatives to rodents in developmental and toxicological studies. These developmental and toxicological studies can be facilitated by the use of transgenic reporters that permit the real-time, noninvasive observation of the fish. Here we report the construction and characterization of transgenic medaka lines expressing green fluorescent protein (GFP) under the control of the zebrafish neurogenin 1 (ngn1) gene promoter. Neurogenin (ngn1) is a helix-loop-helix transcription factor expressed in proliferating neuronal progenitor cells early in neuronal differentiation and plays a crucial role in directing neurogenesis. GFP expression was detected from 24 h post-fertilization until hatching, in a spatial pattern consistent with the previously reported zebrafish ngn1 expression. Temporal expression of the transgene parallels the expression profile of the endogenous medaka ngn1 transcript. Further, we demonstrate that embryos from the transgenic line permit the non-destructive, real-time screening of ngn1 promoter-directed GFP expression in a 96-well format, enabling higher throughput studies of developmental neurotoxicants. This strain has been deposited with and maintained by the National BioResource Project and is available on request (http://www.shigen.nig.ac.jp/medaka/strainDetailAction.do?quickSearch=true&strainId=5660).

Topical exposure of the eyes to the organophosphorus insecticide malathion: lack of visual effects

Concern for toxicity following exposure to organophosphorus insecticides led us to investigate whether topical application of either malathion or malathion mixed in a protein bait as used for aerial spray applications could be toxic to the ocular/visual system. Adult male Long‐Evans rats were either untreated or treated with malathion alone (two drops per day in each eye), bait alone (six drops per day in each eye) or malathion and bait (six drops per day in each eye). The dose levels of malathion alone and malathion and bait were chosen based on pilot work and provided approximately equivalent amounts of active ingredient. The rats were treated 5 days a week for 4 weeks. During the final week of treatment, the rats were implanted surgically with cranial recording electrodes overlying the visual projection area of the cerebral cortex. Visual pattern‐evoked potentials (PEPs) were elicited with vertical sinusoidal gratings at three levels of stimulus spatial frequency (0.08, 0.16 and 0.32 cycles per degree) and three levels of visual contrast (0.15, 0.30 and 0.60). After spectral analysis of the PEP waveforms, the amplitude and phase at the stimulus rate (F1) and the first harmonic (F2) were determined. Although F1 and F2 parameters were influenced significantly by manipulation of the stimulus parameters, no significant differences were observed that could be attributed to treatment with the test substances. In addition, an ophthalmological examination of the eyes and a light microscopic evaluation of ocular tissues, including retina and optic nerve, revealed no treatment‐related lesions. The dose levels used in this study were high—approximately 84000 times the exposure per unit surface area expected from aerial spraying—and yet the visual function of the treated subjects was apparently normal. This study identified no significant toxicological concerns regarding direct ocular contact exposure to malathion.

STP Position Paper: Recommended Best Practices for Sampling, Processing, and Analysis of the Peripheral Nervous System (Nerves and Somatic and Autonomic Ganglia) during Nonclinical Toxicity Studies:

Peripheral nervous system (PNS) toxicity is surveyed inconsistently in nonclinical general toxicity studies. These Society of Toxicologic Pathology “best practice” recommendations are designed to ensure consistent, efficient, and effective sampling, processing, and evaluation of PNS tissues for four different situations encountered during nonclinical general toxicity (screening) and dedicated neurotoxicity studies. For toxicity studies where neurotoxicity is unknown or not anticipated (situation 1), PNS evaluation may be limited to one sensorimotor spinal nerve. If somatic PNS neurotoxicity is suspected (situation 2), analysis minimally should include three spinal nerves, multiple dorsal root ganglia, and a trigeminal ganglion. If autonomic PNS neuropathy is suspected (situation 3), parasympathetic and sympathetic ganglia should be assessed. For dedicated neurotoxicity studies where a neurotoxic effect is expected (situation 4), PNS sampling follows the strategy for situations 2 and/or 3, as dictated by functional or other compound/target-specific data. For all situations, bilateral sampling with unilateral processing is acceptable. For situations 1–3, PNS is processed conventionally (immersion in buffered formalin, paraffin embedding, and hematoxylin and eosin staining). For situation 4 (and situations 2 and 3 if resources and timing permit), perfusion fixation with methanol-free fixative is recommended. Where PNS neurotoxicity is suspected or likely, at least one (situations 2 and 3) or two (situation 4) nerve cross sections should be postfixed with glutaraldehyde and osmium before hard plastic resin embedding; soft plastic embedding is not a suitable substitute for hard plastic. Special methods may be used if warranted to further characterize PNS findings. Initial PNS analysis should be informed, not masked (“blinded”). Institutions may adapt these recommendations to fit their specific programmatic requirements but may need to explain in project documentation the rationale for their chosen PNS sampling, processing, and evaluation strategy.