An Easier Method to Analyze Stereologically the Pig's Hippocampus

Abstract

Most neuro-pathophysiological research involving the hippocampus uses rodents as a nonhuman model, due to the extensive experimental descriptive literature and favorable cost. The findings from the rodent hippocampus may not be generalizable to humans, though, as ontogenetically they have different hippocampal components. While the rodent model has these limitations, the use of nonhuman primates is often not feasible due to economic and ethical considerations. Pigs are a translational alternative due to the anatomic similarity of the hippocampus in humans and pigs. Materials and Methods: Eight pigs’ brains were harvested and then analyzed according to our previously established technique. Five brains were frozen and three were stored in formalin. All eight brains were then sent to an independent histology service, where they were sectioned according to the methodology established by Holm 1994. The slabs were 10 µm with 2.5 cm2 of hippocampus cross-sectional area. Results: The mean total hippocampus volume was 892.84 mm3 ± 198.91 mm3 using Holm’s methodology. The mean number of cells per sample (20X magnification settings) was 9996.75, using automated ImageJ cell counting. Discussion: In this study, the counts of hippocampus cells were divided into two regions of interest: CA1 and CA3. Our results show that the mean number of hippocampus cells observed was 5.75 million and 2.25 million, in the CA1 and CA3 regions respectively. Holm reported 4.12 million cells in the CA1 region and 1.51 million cells in the CA3 region. The results presented here indicate the CA1 and CA3 cell percentages being 23% and 9% respectively, which are similar to the percentages reported by Holm (21% and 12%). Conclusion: These results corroborate previous findings and demonstrate a novel and cost-effective way to study the hippocampus of pigs in translational neurological research.

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An Easier Method to Analyze Stereologically the Pig’s Hippocampus
Thiago Bassi1*
, Elizabeth Rohrs 2, Karl Fernandez 2, Marlena Ornowska 2 and Steven Reynolds2
1 Research and Development at Lungpacer Medical Inc. Vancouver, Canada
2 Fraser Health, Vancouver Canada
*Corresponding Author : Thiago Bassi, Research and Development at Lungpacer Medical Inc. Vancouver, Canada.
E-mail: tbassi@lungpacer.com
Received date: July 17, 2019; Accepted date : July 23, 2019; Published date: July 25, 2019
Citation : Bassi T, Rohrs E, Fernandez K, Ornowska M, and Reynolds S. (2019) An Easier Method to Analyze Stereologically the Pig’s
Hippocampus. J Neuroscience and Neurological Surgery. 4(3): Doi: 10.31579/2578-8868 /078
Copyright : © 2019 Thiago Bassi. This is an open-access article distributed under the terms of The Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Introduction
Most neuro-pathophysiological research involving the hippocampus
uses rodents as a nonhuman model, due to the extensive experimental
descriptive literature and favorable cost. The findings from the rodent
hippocampus may not be generalizable to humans, though, as
ontogenetically they have different hippocampal components.
Moreover, the hippocampus proportions and architectonics are
significantly different between rodents and humans. While the rodent
model has these limitations, the use of nonhuman primates is often not
feasible due to economic and ethical considerations. Pigs are a
translational alternative due to the anatomic similarity of the
hippocampus in humans and pigs. In addition, pigs are a well-accepted
animal research model and have lower cost when compared to
nonhuman primates.
Human-size pigs have been used in research due to their anatomical
and physiological similarity to humans. [1,2] The pig’s brain has a
gyrencephalus similar to humans, which facilitates surgical procedures
and interventions. Pigs are genetically relatively homogeneous, similar
to inbred laboratory rats, leading to a minimal interindividual
variation.
A publication by Holm in 1994 provided a quantitative description of
fundamental structural parameters, regional volumes and neuron
numbers in the hippocampus of the domestic pig [3]. The volumetric
and numerical data presented by Holm provided a unique opportunity
to evaluate structural differences in homologous hippocampus areas
between pigs and humans, and obtain a better understanding of the
functional consequences of the differences in size. Despite this strong
foundation, the pig hippocampus has not been clearly established as
the translational model of choice for neurological studies.
In 2016, van Dijk published a systematic review comparing the
hippocampus in 18 different species, where one pig study was reported
[4].
The hippocampus is well recognized as the hub of neuroplasticity and
neurogenesis in the central nervous system. Many diseases begin in areas
CA1 and CA3 before spreading to the cortex. As an example, Masurkar,
2018 explains in great detail the progression of Alzheimer’s disease,
where the CA1 area is the starting point [5]. Recently, there has been an
increased interest in derangements in the CA1 and CA3 areas as the basis
for important clinical syndromes such as memory impairment and spatial
recognition deficit [5]. Historically, many researchers have been using
rodents to analyze the hippocampus, but this poses significant translational
limitations, as discussed above.
Herein, we report work that can serve as a basis for pig-based
neuroscience research. The aim of this study is to extend previous work by
Holm with a focus on CA1 and CA3 volume and cell count
Materials and methods
Animal Care and Ethical approvals were obtained, and post-euthanasia
surgical procedures were conducted at UBC Research Centre in
Vancouver, Canada. Eight pigs' brains were harvested and then analyzed
according to our previously established technique [6]. Five brains were
frozen and three were stored in formalin. All eight brains were then sent to
an independent histology service, where they were sectioned according to
the methodology established by Holm 1994. The slabs were 10 µm with
2.5
cm2 of hippocampus cross-sectional area. The hippocampus cell
calculations were made according to Holm’s formula:
Hippocampus Cells (N)=Cells per slab*2500
Abstract
Most neuro-pathophysiological research involving the hippocampus uses rodents as a nonhuman model, due to the extensive experimental
descriptive literature and favorable cost. The findings from the rodent hippocampus may not be generalizable to humans, though, as ontogenetically
they have different hippocampal components. While the rodent model has these limitations, the use of nonhuman primates is often not feasible due
to economic and ethical considerations. Pigs are a translational alternative due to the anatomic similarity of the hippocampus in humans and pigs.
Materials and Methods: Eight pigs' brains were harvested and then analyzed according to our previously established technique. Five brains were
frozen and three were stored in formalin. All eight brains were then sent to an independent histology service, where they were sectioned according
to the methodology established by Holm 1994. The slabs were 10 µm with 2.5 cm2 of hippocampus cross-sectional area. Results: The mean total
hippocampus volume was 892.84 mm3 ± 198.91 mm3 using Holm’s methodology. The mean number of cells per sample (20X magnification
settings) was 9996.75, using automated Image J cell counting. Discussion: In this study, the counts of hippocampus cells were divided into two
regions of interest: CA1 and CA3. Our results show that the mean number of hippocampus cells observed was 5.75 million and 2.25 million, in the
CA1 and CA3 regions respectively. Holm reported 4.12 million cells in the CA1 region and 1.51 million cells in the CA3 region. The results
presented here indicate the CA1 and CA3 cell percentages being 23% and 9% respectively, which are similar to the percentages reported by Holm
(21% and 12%). Conclusion: These results corroborate previous findings and demonstrate a novel and cost-effective way to study the hippocampus
of pigs in translational neurological research.
Keywords: neuroscience; stereological method; hippocampus; pigs
Abbreviations: CA-Cornus Ammonis
Open Access
Research Article
Journal of Neuroscience and Neurological Surgery
Thiago Bassi
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According to the previous work by Holm, the formula above was
determined using the number of cells per slab multiplied by the size of
the hippocampus extension. To replicate this work, the ITCN ImageJ
software plugin was used to count the number of cells per slab. Next,
the ImageJ ruler was used to measure the area of the hippocampus.
Holm’s formula was applied to obtain the final number of
hippocampal cells per subject per area. Finally, the CA1 and CA3
percentage were calculated applying the same formula on the
respective areas (Figure 1).
Figure1: Hippocampus measurements using ImageJ. Black ellipses
represent two hippocampus areas of interest.
Results and Discussion
The mean total hippocampus volume was 892.84 mm3 ± 198.91 mm3
using Holm’s methodology. The mean number of cells per sample
(20X magnification settings) was 9996.75, using automated ImageJ
cell counting. The percentages of hippocampal cells in areas CA1 and
CA3 were 9% and 23% respectively, after normalizing by weight.
Results are listed in table 1.
Hippocampus Volume
892.84mm³±198.91
mm³
Mean Number of Cells per sample (20X
magnification settings)
9996.75
Mean Number of Total Hippocampus
cells counted per subject
24.99 million
CA1 Mean Number of cells per subject
5.75 million
CA3 Mean Number of cells per subject
2.25 million
CA1 Mean cell percentage per subject
23%
CA3 Mean cell percentage per subject
9%
Table 1. Results
The cytoarchitecture of the pig hippocampus is similar to humans.
Thus, it is crucial to establish a stereological number of hippocampal
cells in pigs as a basis for future studies.
It is important to correct the cell counts in proportion to body weight,
as we did, and as Holm did.
Our results showed an average number of hippocampus cells of 24.99
million. This is similar to those reported by Holm (21.61 million).
In this study, the counts of hippocampus cells were divided into two
regions of interest: CA1 and CA3.
Our results show that the mean number of hippocampus cells observed
was 5.75 million and 2.25 million, in the CA1 and CA3 regions
respectively. These results are comparable to previous studies. Holm
reported 4.12 million cells in the CA1 region and 1.51 million cells in the
CA3 region. The results presented here indicate the CA1 and CA3 cell
percentages being 23% and 9% respectively, which are similar to the
percentages reported by Holm (21% and 12%). It is important to highlight
that the proportion of cells is similar in the human hippocampus.
This study provides pig hippocampus data to be used in future studies. So
far, only one paper was found to address this topic. [4] However, the use
of open source software to stereologically study the hippocampus was not
mentioned as part of the reported methodology. The use of open source
software (in our case, ImageJ) is a novel and cheaper approach to
stereologically analyze the brain tissue.
Limitations: On this study we did not analyze pigs' diets, which might
change the hippocampus structure and volume [7] being a variable to be
considered in future comparative studies.
Conclusion
These results corroborate previous findings and demonstrate a novel and
cost-effective way to study the hippocampus of pigs in translational
neurological research.
Acknowledgements
This study was funded by Lungpacer Medical Inc.
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