Introduction Conclusion

Figure 1: Piriform Cortex Cellular Comparison AbstractKainic Acid is commonly used to induce recurrent seizures

in animal model experimentation. The consequence is selective limbic system damage and neuron degradation. Kainic acid is a non-NMDA receptor agonist, and is 30-fold more toxically potent than glutamate. (Zhang and Zhu) Glutamate excitotoxicity is triggered by an excessive influx of extracellular calcium, occurring after overstimulation of a glutamate receptor. Excitotoxicity from recurrent glutamate overstimulation is behind the neuronal pathology of diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Kainic Acid (KA) acts upon ionotropic (fast) glutamate receptors, which initiates postsynaptic potentials directly to ion channels. Kainic acid will bind, with greater affinity than glutamate, to Gluk4 and Gluk5. These genes are synonymous with kainate, a glutamate receptor subtype family.

Kainic Acid’s successful binding to these receptors is known to cause brain damage and extensive central nervous system (CNS) pathology. Necrosis or cell death can be observed after status epilepticus is initiated post KA injection intra-peritoneally (IP). Neuro pathological assessment of brain regions effected will be wide spread through limbic affiliated structures such as Piriform/Entorhinal Cortex (EC), Hippocampal fields (CA1, CA2, CA3), and Dentate Gyrus which receives neuronal input from the EC and projects to CA3.

A Special Thank You to the Family of FRANK P. PALOPOLI

(Inventor of the fertility drug, Clomid)Thanks for the support!

Gregory Wright• Julia Paulus • JaQuay Wheatley. • David Zuzga Ph. D. • Gerald Ballough Ph.D.

Consequential Cerebral Insult Via Kainic Acid Exposure: Assessing Pathological Variation in Glutamate Excitotoxic Models

Department of Biology, La Salle University, Philadelphia, Pennsylvania

Results

Figure 1: Each picture is recorded from 4ųm coronal sections of non-treatment control, or kainic acid experimental rattus norvegicus cerebrum. Slides A, B, and C show control piriform cortex at low magnification in three different stains, Hematoxylin and Eosin (H&E) (A), Bielschowsky Silver (B), and Fluoro-Jade B (C). A and B show the left piriform cortex where as C displays the right. Although the Hematoxylin and Eosin (H&E) staining in figure A displays darkly stained neurons(blue arrows), these are only “Dark Neuron” artifacts consequential from some H&E methods. Picture D contains the typical healthy neurons of control piriform cortex at high magnification. The high mag. control pictures (D, E, and F) display neurons from controls’ in each of the three different stains. In these higher magnifications of the controls, the Bielschowsky silver nitrate staining method (E) better distinguishes nuclei than H&E (D). The cerebral sections, from kainic acid exposed rats, in the second row, display the spongiform cellular morphology (G, H), and positive Fluoro-Jade B (I) indicative of neuronal degeneration in the experimental rat cerebrum. These features are highlighted in the high magnification in the third row (J,K, and L). The picture in (C) demonstrates the expected lack of positive fluorescence in the piriform, below it, (I) is a piriform from a rat exposed to IP injection of kainic acid. (L) shows the highly magnified positive neurons which express degeneration via fluorescence, as well as degenerative neuron swelling/shrinkage. Eosinophilic “red dead” neurons can be observed in slide J (arrows), where the neurons’ cytoplasm stains densely with eosin and the nucleus condenses to demonstrate pyknosis and thus cellular injury. Nuclear condensation or pyknosis also occurs in slide K (arrows), where two neurons have cause extracellular vacuolation due to excessive swelling/shrinking. Staining; H&E (A, D, G, J), Bielschowsky (B, E, H, K), Fluoro-jade B (C, F, I, L) Final Mag.; 100x( C, I), 400x (A, B, G, H), 600x (F, L), 1000x (D, E, J, K)

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Hematoxylin & Eosin Bielschowsky’s Fluoro-Jade B

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Neuro pathological assessments can prove extremely difficult, but there are multiple ways to assess neuron pathology using tissue staining and light microscopy. The damage present in the CNS after kainic acid injection, should overlay most of the limbic system brain regions. In this study, the piriform cortex is chosen to be the limbic region of assessment because of its connections with other major limbic regions. H&E neuron assessment can show consequential damages to cellular nuclei and surrounding matrix. Pyknotic (chromatin condensation), karyorrhectic (nuclear fragmentation), and brightly stained eosinophilic cytoplasm all are irrefutable signs of cellular damage and pending cellular death. Although the mechanism is yet to be defined, fluoro-jade B positive cellular fluorescence is evidence of degeneration and is accepted as a marker for cell death. Bielschowsky’s silver is specific for neuronal fibers, axons and neurofibrillary tangles; which will show detailed cytoarchitecture compared to the most common method of staining, H&E. By viewing a control piriform cortex along with a cerebrum exposed to the excitotoxic effects of kainate, there should be a clear culmination of cellular injury due to the widespread necrotic cell death affiliated with glutamate excitotoxic seizures.

Nuclear Pyknosis can be seen in picture K, under high magnification of Bielschowsky staining. Also high magnification H&E displays eosinophilic “red dead” cytoplasm, indicative of neuronal degeneration. Excessive swelling and shrinking of neurons, post processing, can be seen in pictures G, H, I, K, and L are often closely associated with neuronal damage. Positive Fluoro-Jade B fluorescence is present throughout piriform (I), where brightly stained eosinophilic cells are present as well (G, J). The lower magnification pictures show damage is present throughout piriform toward perirhinal cortex (G, H, I). The non-treated control rat displays normal piriform histology, without fluoro-jade fluorescence or excessive vacuolation.

Definitive Neuronal Degeneration has been uncovered from the Kainic Acid Injections. The Fluoro-jade B method of staining is highlighting neurons in areas of significant pathology shown by the congruent H&E and Bielschowsky stains with sufficient cytoplasmic eosinophilia and pyknosis. Not only has damage been demonstrated through nuclear changes but we have observed morphological change (spongiform appearance) in an entire brain region, the piriform. A comparative look at the limbic accessory region, the piriform, proves our lab is capable of accessing irreversible neuro pathology via IP injection of a known glutamate excitotoxic molecule, while also identifying histological neuronal artifacts similar to dead neurons. These methods, along with the other data collection from the Veritas team members, should be able to produce similar results with other neuro toxic molecules (I.e. ethanol in binge exposure), so that our lab can further analyze the depth of neuronal insult in all brain regions, with sound knowledge of “bona fide” neuronal cell death analysis.

Materials/MethodsHilltop lab animals, Inc. (Scottsdale, PA) provided the

rats for experimentation. The animals were kept alive for 8 days prior to deliverance of kainic acid (12mg/kg) to the experimental group, and were euthanized the next day after grand mal seizures had occurred. Tissues were cleared of blood with 10% buffered formalin via transcardial perfusion. All injections were done IP. Tissues were embedded with paraffin wax after processing, and a microtome was used to section tissues at 4ųm, and all stained sections were taken near bregma -3.30.