Germán Santamarina

β-amyloid cortical deposits are accompanied by the loss of serotonergic neurons in the dog

Dogs may naturally suffer an age‐related cognitive impairment that has aroused a great deal of interest, even beyond the field of the veterinary clinic. This canine senile dementia reproduces several key aspects of Alzheimers disease (AD), including the presence of β‐amyloid (Aβ) deposits in the cerebral cortex, neurodegeneration, and learning and memory impairments. In the present study, we have used unbiased stereological procedures to estimate the number of the dorsal and median raphe nuclei (DRN and MRN, respectively) serotonergic neurons immunolabeled with an anti‐tryptophan hydroxylase (TrH) monoclonal antibody in young and aged dogs without Aβ cortical deposits and in aged dogs with Aβ cortical deposits. The estimated total number of TrH‐labeled neurons (mean ± SD) was 94,790 ± 26,341 for the DRN and 40,404 ± 8,692 for the MRN. The statistical analyses revealed that aged dogs with Aβ cortical pathology had 33% fewer serotonergic neurons in the DRN and MRN than aged dogs without Aβ cortical deposits (108,043 ± 18,800 vs. 162,242 ± 39,942, respectively; P = 0.01). In contrast, no significant variations were found between young and aged dogs without Aβ cortical deposits. These results suggest that degeneration of the serotonergic neurons could be involved in the cognitive damage that accompanies Aβ cortical pathology in the dog and reinforce the use of the canine model for exploring the potential mechanisms linking the cortical Aβ pathology and serotonergic neurodegeneration that occurs during the course of AD. J. Comp. Neurol. 513:417–429, 2009.

Dogs with canine counterpart of Alzheimer's disease lose noradrenergic neurons.

Degeneration of noradrenergic neurons in the locus ceruleus is a well-described feature of Alzheimers disease (AD). In spite of extensive utilization of the dog as a model for human degenerative diseases, there is no data on the response to aging of the noradrenergic system in dogs. We have used modern unbiased stereology to estimate the total number of A6-A7 noradrenergic neurons in normal, aged dogs and dogs with the canine counterpart of AD. In small-breed dogs with no cognitive impairments, the total mean number of tyrosine hydroxylase immunolabeled A6-A7 neurons was 17,228+/-1655, with no differences between young and aged dogs. In contrast, aged dogs with cognitive impairments exhibited a significant reduction in the total number of A6-A7 neurons (13,487+/-1374; P=0.001). Additionally, we found a negative correlation between the number of A6-A7 neurons and the extent of beta-amyloid deposits in the prefrontal cortex. These results suggest that the canine model could be useful in exploring the potential benefits of noradrenergic drugs for the treatment of AD.

Plasma β-amyloid peptides in canine aging and cognitive dysfunction as a model of Alzheimer's disease

Aging dogs naturally demonstrate cognitive impairment and neuropathology that model early Alzheimers disease (AD). In particular, there is evidence that canine cognitive dysfunction syndrome (CDS) in aged dogs is accompanied by cortical deposition of Aβ peptides and neurodegeneration. Plasma Aβ levels have been examined in humans as putative biomarkers for AD, but to date, no similar studies have been conducted for canine dementia. The aim of the present study was to assess plasma Aβ1-42 and Aβ1-40 levels in a blind study using pet dogs that were either successfully aging or exhibiting CDS. The severity of cognitive impairment was assessed using an owner-based questionnaire. On average, young dogs presented significantly higher plasma levels of Aβ1-42 and Aβ1-40 than aged, cognitively unimpaired dogs. Notably, among aged dogs, the levels of Aβ1-42 and the Aβ42/40 ratio were significantly higher in those showing mild cognitive impairment than in either cognitively unimpaired or severely affected dogs. These results suggest that increased plasma Aβ1-42 levels and Aβ42/40 ratio could be a biomarker for canine cognitive dysfunction, which is considered an excellent natural model of early AD.

ELECTROCARDIOGRAM REFERENCE VALUES FOR THE BUZZARD IN SPAIN

Electrocardiographic reference values were established on apparently healthy buzzards (Buteo buteo) in Lugo (Spain) from March 1997 to June 1999. All birds were anesthetized with isofluorane and placed in dorsal recumbence. The standard and augmented unipolar limb leads electrocardiograms were recorded in 65 buzzards. The wave forms were analyzed in lead II at 50 mm/sec and at 1 cm = 1 mV to determine P, PR, QRS, T and QT durations and P, QRS and T amplitudes. The polarity of each wave form was tabulated in all leads. The mean electrical axis (MEA) for the frontal plane was calculated using leads II and III. The mean heart rate was 325.2 ± 52.9 beats/min. In lead II, the P wave was positive, the dominant pattern of QRS complex was QS and the T wave was always positive. The average value of the MEA was −99.2 ± 7.7°. Establishment of normal electrocardiogram (EKG) values will facilitate a better understanding of EKG changes seen in many diseases of these birds.

In vivo arrhythmogenicity of the marine biotoxin azaspiracid-2 in rats

Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellate Azadinium spinosum that accumulate in several shellfish species. Azaspiracid poisoning episodes have been described in humans due to ingestion of AZA-contaminated seafood. Therefore, the contents of AZA-1, AZA-2 and AZA-3, the best-known analogs of the group, in shellfish destined to human consumption have been regulated by food safety authorities of many countries to protect human health. In vivo and in vitro toxicological studies have described effects of AZAs at different cellular levels and on several organs, however, AZA target remains unknown. Very recently, AZAs have been demonstrated to block the hERG cardiac potassium channel. In this study, we explored the potential cardiotoxicity of AZA-2 in vivo. The effects of AZA-2 on rat electrocardiogram (ECG) and cardiac biomarkers were evaluated for cardiotoxicity signs besides corroborating the hERG-blocking activity of AZA-2. Our results demonstrated that AZA-2 does not induce QT interval prolongation on rat ECGs in vivo, in spite of being an in vitro blocker of the hERG cardiac potassium channel. However, AZA-2 alters the heart electrical activity causing prolongation of PR intervals and the appearance of arrhythmias. More studies will be needed to clarify the mechanism by which AZA-2 causes these ECG alterations; however, the potential cardiotoxicity of AZAs demonstrated in this in vivo study should be taken into consideration when evaluating the possible threat that these toxins pose to human health, mainly for individuals with pre-existing cardiovascular disease when regulated toxin limits are exceeded.

Effect of age and severity of cognitive dysfunction on two simple tasks in pet dogs

Dogs exhibit age-dependent losses in learning and memory as well as a progressive accumulation of neuropathology that parallels that observed in normal human aging and early Alzheimers disease. These deficits have been extensively studied using a number of standard cognitive tasks in the laboratory; however, appropriate tools for their assessment in veterinary clinics are still lacking. The aim of this study was to evaluate the effect of age and the severity of cognitive dysfunction syndrome (CDS) on two simple tests conducted in a clinical setting. A food searching (FS) task and a problem-solving (PS) task were administered to young (1-4 years, n=9), middle-aged (5-8 years, n=10), cognitively unimpaired aged (≥9 years, n=31), and cognitively impaired aged (≥ 9 years, n=37) dogs. Cognitive status was classified using an owner-based questionnaire, and in the impaired group, dogs were categorized as having either mild or severe CDS. During the FS task, younger dogs (<9 years) were able to locate the food more quickly and with more success than the aged groups (≥9 years). Dogs with severe CDS exhibited poorer performance than those with mild CDS or their healthy counterparts. In the PS task, younger dogs performed better than the aged dogs in obtaining food, but there were no differences related to CDS severity. The FS task might help to better characterize cognitively affected dogs in the clinical setting than the PS task. These and similar tasks require further investigations in the field.

Acute Cardiotoxicity Evaluation of the Marine Biotoxins OA, DTX-1 and YTX

Phycotoxins are marine toxins produced by phytoplankton that can get accumulated in filter feeding shellfish. Human intoxication episodes occur due to contaminated seafood consumption. Okadaic acid (OA) and dynophysistoxins (DTXs) are phycotoxins responsible for a severe gastrointestinal syndrome called diarrheic shellfish poisoning (DSP). Yessotoxins (YTXs) are marine toxins initially included in the DSP class but currently classified as a separated group. Food safety authorities from several countries have regulated the content of DSPs and YTXs in shellfish to protect human health. In mice, OA and YTX have been associated with ultrastructural heart damage in vivo. Therefore, this study explored the potential of OA, DTX-1 and YTX to cause acute heart toxicity. Cardiotoxicity was evaluated in vitro by measuring hERG (human èter-a-go-go gene) channel activity and in vivo using electrocardiogram (ECG) recordings and cardiac damage biomarkers. The results demonstrated that these toxins do not exert acute effects on hERG channel activity. Additionally, in vivo experiments showed that these compounds do not alter cardiac biomarkers and ECG in rats acutely. Despite the ultrastructural damage to the heart reported for these toxins, no acute alterations of heart function have been detected in vivo, suggesting a functional compensation in the short term.

Effect of age and severity of cognitive dysfunction on spontaneous activity in pet dogs – Part 2: Social responsiveness

Changes in social interactions with owners and other dogs are frequently observed in dogs with cognitive dysfunction syndrome (CDS). The aim of this work was to assess the effect of age and severity of CDS on social responsiveness. This is the second part of a 2-part report on spontaneous activity in pet dogs. A human interaction test and a mirror test were administered at baseline and 6 months later to assess social responses to humans and conspecifics, respectively, to four groups of privately-owned dogs: young (n=9), middle-aged (n=9), cognitively unimpaired aged (n=31), and cognitively impaired aged (n=36). The severity of cognitive impairment was considered in the last group and dogs were categorised as having either mild or severe CDS. The influence of the person and the mirror on locomotion and exploratory behaviour was also studied. Dogs were recorded in a testing room and the video recordings were subsequently analysed. Young dogs displayed more interactions involving physical contact with a person. Young and middle-aged dogs showed more vocalisations in response to social isolation. In contrast, aged animals spent more time in front of the mirror. Changes in social responsiveness associated with severe CDS included decreased response to social isolation and human interaction and increased time in front of the mirror, suggesting a deficit in habituation. Testing of spontaneous activity might help to characterise CDS in aged dogs, a condition increasingly diagnosed in veterinary clinics and a potentially useful natural model of Alzheimers disease in humans.

Effect of age and severity of cognitive dysfunction on spontaneous activity in pet dogs - Part 1: Locomotor and exploratory behaviour

Age-related cognitive dysfunction syndrome (CDS) has been reported in dogs and it is considered a natural model for Alzheimers disease in humans. Changes in spontaneous activity (including locomotor and exploratory behaviour) and social responsiveness have been related to the age and cognitive status of kennel-reared Beagle dogs. The aim of this study was to assess the influence of age and severity of CDS on locomotor and exploratory behaviour of privately owned dogs. This is the first part of a two-part report on spontaneous activity in pet dogs. An open-field (OF) test and a curiosity test were administered at baseline and 6 months later to young (1-4 years, n=9), middle-aged (5-8 years, n=9), cognitively unimpaired aged (≥ 9 years, n=31), and cognitively impaired aged ( ≥ 9 years, n=36) animals. Classification of cognitive status was carried out using an owner-based observational questionnaire, and in the cognitively impaired group, the dogs were categorised as having either mild or severe cognitive impairment. Dogs were recorded during sessions in the testing room and the video-recordings were subsequently analysed. The severity of CDS (but not age) influenced locomotion and exploratory behaviour so that the more severe the impairment, the higher the locomotor activity and frequency of corner-directed (aimless) behaviours, and the lower the frequency of door-aimed activities. Curiosity directed toward novel stimuli exhibited an age-dependent decline although severely affected animals displayed more sniffing episodes directed towards the objects. OF activity did not change after 6 months. Testing aged pet dogs for spontaneous behaviour might help to better characterise cognitively affected individuals.

Subacute Cardiovascular Toxicity of the Marine Phycotoxin Azaspiracid-1 in Rats

Azaspiracids (AZAs) are marine toxins produced by Azadinium spinosum that get accumulated in filter feeding shellfish through the food-web. The first intoxication was described in The Netherlands in 1990, and since then several episodes have been reported worldwide. Azaspiracid-1, AZA-2, and AZA-3 presence in shellfish is regulated by food safety authorities of several countries to protect human health. Azaspiracids have been related to widespread organ damage, tumorogenic properties and acute heart rhythm alterations in vivo but the mechanism of action remains unknown. Azaspiracid toxicity kinetics in vivo and in vitro suggests accumulative effects. We studied subacute cardiotoxicity in vivo after repeated exposure to AZA-1 by evaluation of the ECG, arterial blood pressure, plasmatic heart damage biomarkers, and myocardium structure and ultrastructure. Our results showed that four administrations of AZA-1 along 15 days caused functional signs of heart failure and structural heart alterations in rats at doses ranging from 1 to 55 µg/kg. Azaspiracid-1 altered arterial blood pressure, tissue inhibitors of metalloproteinase-1 plasma levels, heart collagen deposition, and ultrastructure of the myocardium. Overall, these data indicate that repeated exposure to low amounts of AZA-1 causes cardiotoxicity, at doses that do not induce signs of other organic system toxicity. Remarkably, human exposure to AZAs considering current regulatory limits of these toxins may be dangerously close to clearly cardiotoxic doses in rats. These findings should be considered when human risk is estimated particularly in high cardiovascular risk subpopulations.