Cell injury/death leads to N/A, Neurodegeneration

There is well established mechanistic understanding supporting the relationship between these two KEs.

Neurodegeneration in the strict sense of the word, is referring to any pathological condition primarily affecting brain cell populations (Przedborski et al., 2003). At histopathological level, neurodegenerative conditions are described by neuronal death and reactive gliosis (Przedborski et al., 2003).

Include consideration of temporal concordance here

• Acute brain damage induced by DomA is characterized by neurodegenerative changes consisting of neuronal shrinkage, vacuolization of the cytoplasm, cell drop out, edema, microvacuolation of the neuropil and hydropic cytoplasmic swelling of resident astrocytes (reviewed in Pulido et al., 2008). These histopathological changes can be identified within structures of the limbic system, in hippocampus, in the CA3, CA4 or hilus of the dentate gyrus (DG) (reviewed in Pulido et al., 2008). Other brain areas known to be affected by DomA include: the olfactory bulb, the piriform and entorhinal cortices, the lateral septum, the subiculum, the arcuate nucleus and several amygdaloid nuclei. The area postrema is another target for DomA toxicity as it has been identified in both rodents and non-human primates, providing a possible explanation of emetic symptoms (nausea, retching, and/or vomiting) induced by DomA. There has been an effort to map and create a 3-D reconstruction of DomA-induced neurodegeneration in the mouse brain demonstrating that the affected areas include the olfactory bulb, septal areas and the limbic system (Colman et al., 2005; Barlow et al., 2004).

• Female Sprague-Dawley rats dosed once intraperitoneally (i.p.) with 0, 1, 2, 4, or 7.5 DomA mg /kg of body weight have been euthanized after 24 h and their nervous system has been examined for microscopic alterations revealing neuronal degeneration and vacuolation of the neurophil in the limbic and the olfactory systems (Tryphonas et al., 1990).

• The mean of TUNEL positive cells in the hippocampus is increased (6 fold) in mice injected intraperitoneally (i.p.) at a dose of 2 DomA mg/kg once a day for 3 weeks (Lu et al., 2012). However, the same treatment protocol does not cause any neurodegeneration (Lu et al., 2012). In contrast, when the same treatment has been prolonged for one more week (total 4 weeks), the mean values of NeuN-positive cells in the hippocampal CA1 sections of DomA-treated cells decreases by 3 fold compared to controls (Lu et al., 2012). This study shows that the incidence of upstream KE (cell death) is higher than the incidence of downstream KE (neurodegeneration) and that upstream KE (cell death) precedes downstream KE (neurodegeneration).

• The bcl-2 and bax mRNA levels in the hippocampus are significantly increased at 16 h and gradually decreased at 24 h following the administration of DomA (0.75 mg/kg body weight) in adult rats. In situ hybridization analysis reveals complete loss of bcl-2, bax, and caspase-3 mRNA at 24 h after DA administration in the region of the hippocampus, whereas neurodegeneration by Nissl staining is detected at the same time point but has been reported to be more pronounced after 5 days (Ananth et al., 2001). This study demonstrates that both KEs occur after exposure to the same dose of DomA and that the upstream KE (cell death) occurs earlier than the downstream KE (neurodegeneration).

• Adult rats received i.p. injections with DomA 1.0 mg/kg/h until animals exhibited first motor seizures. After a week of recovery, aggressive behaviors and motor seizures of the animals have been monitored for 3h twice a week. After 12 weeks, animals were euthanized and brains have been examined for indications of cell loss by using thionine (Nissl) staining, which highlights the cell bodies of all living neurons. In piriform cortex a reduced cell density has been noted in the medial layer 3 (1.3-1.8 fold decrease compared to controls), an area that shows also prominent amino cupric staining (stain that assesses neuronal damage) (Tiedeken and Ramsdell, 2013a). The same research group has reported that by following the above experimental procedure but sacrificing the rats 7 days after DomA-induced seizures intense and widespread silver reaction product in the olfactory bulb occurs, whereas minor or no evident damage is found in the hippocampus (Tiedeken et al., 2013b).

• Injection of DomA 0.5 mg/kg, i.p. to adult C57BL/6 male mice resultes in loss of 32% and 30% of Nissl-stained neurons in hilus and CA1 pyramidal layer of the hippocampus, respectively, compared to control mice when they are sacrificed 7 d after the administration (Antequera et al., 2012).

• The severity and extent of hippocampal neuronal degeneration varies significantly depending on the dose of DomA (1 μM to 1 mM) that is tested after microinjection to adult male Sprague Dawley rats (Qiu and Currás-Collazo, 2006). In rats dosed with 1 mM DomA and sacrificed after 24 h, histopathological analysis using toluidine blue staining has revealed extensive neuronal damage throughout the ipsilateral hippocampal structure. Shrunken, disorganized and densely stained neurons of irregular shape have been identified throughout CA1, CA2, CA3 pyramidal layer as well as the dentate gyrus hilus and granule cells layer. For the 100 μM group animals, CA1 neuronal changes have been less prominent, whereas 10 μM and 1 μM DomA have not produced any resolvable histopathological changes (Qiu and Currás-Collazo, 2006).

• Adult male rats treated with 2 mg/kg DomA i.p. have been sacrificed after 3 d and showed that the silver stain that is used to assess neurodegeneration clearly distinguishes treated from control animals, whereas a number of other markers has failed to do so (Scallet et al., 2005). The same results have been found after even longer exposure times (7 d) to DomA (Appel et al., 1997).

• Male Wistar rats have been given a single i.v. injection of DA (0.75 mg/kg) in the right external jugular vein and brain sections have been stained with Nissl stain at 5 d after DomA administration. Histopathological analysis has revealed a large number of darkly stained shrunken neurons in the hippocampus (Ananth et al., 2003). However, complete absence of hippocampal neurons has been observed in CA1 and CA3 regions in DomA treated animals at 3 months after DomA administration (Ananth et al., 2003).

• In 2-3 week old hippocampal slice cultures, derived from 7 day old rat pups, DomA (0.1-100 µM) has been added to the culture medium and neurodegeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields has been measured. The CA1 region appears to be most sensitive to DomA, with an EC50 value of 6 µM DomA after estimating the PI-uptake at 72 h (Jakobsen et al., 2002).

• Cynomolgus monkeys have been given i.v. a range of DomA doses from 0.25 to 4.0 mg/kg. Silver staining of brain sections have revealed that doses in the range of 0.5-1.0 mg/kg produces a small area of silver grains restricted to axons of the hippocampal CA2 stratum lucidum, whereas higher concentrations produce degenerating axons and cell bodies (Slikker et al., 1998). The same research group treated i.v. adult monkeys with DomA at one of a range of doses from 0.25 to 4 mg/kg. After a week, silver staining has demonstrated degenerating axons and cell bodies that is mild and restricted to CA2 stratum lucidum at a lower doses (0.5 to 1.0 DomA mg/kg). Doses of more than 1.0 mg/kg cause widespread damage to pyramidal neurons and axon terminals of CA4, CA3, CA2, CA1, and subiculum subfields of the hippocampus. However, when DomA is orally administered to cynomolgus monkeys at doses of 0.5 mg/kg for 15 days and then at 0.75 mg/kg for another 15 days no histopathoogical changes in the brain are detected (Truelove et al., 1997).

• In humans, autopsy of individuals intoxicated by DomA reveal brain damage characterized by neuronal necrosis and in the hippocampus and the amygdaloid nucleus (Pulido, 2008). The thalamus and subfrontal cortex are damaged only in some patients suffering from Amnesic Shellfish Poisoning (ASP). The detailed examination of one patient intoxicated by DomA has revealed complete neuronal loss in the CA1, CA3 and CA4 regions, whereas moderate loss is seen in the CA2 region (Cendes et al., 1995). Non-severe neuronal loss has been detected in amygdale, overlying cortex, the dorsal and ventral septal nuclei, the secondary olfactory areas, and the nucleus accumbens (Cendes et al., 1995).

Gap of knowledge: there are no studies showing that GLF-induced cell death leads to neurodegeneration.

Zebrafish has been exposed for 36-weeks to DomA and has showed no excitotoxic neuronal death and no histopathological lesions in glutamate-rich brain areas (Hiolski et al., 2014).

Administration of DomA (9.0 mg DomA kg(-1) bw, i.p.) to Sparus aurata (seabream) leads to measurement of 0.61, 0.96, and 0.36 mg DomA kg(-1) of brain tissue at 1, 2 and 4 hours. At this dose but also at lower concentrations (0.45 and 0.9 mg DomA kg(-1) bw) no major permanent brain damage has been detected (Nogueira et al., 2010). Leopard sharks possess the molecular target for DomA but it has been shown to be resistant to doses of DomA that can cause neurotoxicity to other vertebrates, suggesting the presence of some protective mechanism (Schaffer et al., 2006).

All these reports support the view that there is possible a species specific susceptibility to DomA toxicity.