- Email: [email protected]
The temporal pattern of postnatal neurogenesis found in the neocortex of the Göttingen minipig brain
Neuroscience 195 (2011) 176 –179
THE TEMPORAL PATTERN OF POSTNATAL NEUROGENESIS FOUND IN THE NEOCORTEX OF THE GÖTTINGEN MINIPIG BRAIN J. HOU,* N. ERIKSEN AND B. PAKKENBERG
Abstract—The Göttingen minipig (G-mini) is increasingly used as a non-primate model for human neurological diseases. We applied design-based stereology on five groups of G-minis aged 1 day, 14 days, 30 days, 100 days, and 2 years or older to estimate the pattern of postnatal neuron number development in the neocortex. Two time periods for the postnatal increase of neocortical neuron number were observed from the time of birth to day 14 (Pⴝ0.013) and from day 30 to day 100 (P<0.001). No significant change in neuron number was found from day 14to 30 (Pⴝ0.58) and day 100 onward (Pⴝ0.39). The average estimated total number of neurons in the neocortex was 236, 274, 264, 338, and 353 million, respectively. Since neurogenesis and neuronal migration in the human neocortex are generally accepted to be complete before term, the application of G-mini as human disease models may be inappropriate before day 100. However, G-mini may serve as a valuable model for the studies of ongoing neurogenesis in the living brain. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
chi et al., 1994; Sachs, 1994). In recent years, G-mini is winning increasing popularity in neuroscience, as it reflects the geometry and complexity of the cytoarchitecture found in the human CNS, while at the same time circumvents the technical and organizational difficulties and high costs related to non-human primate models. Recently, with the development of transgenic and chemically induced models of neurodegenerative disorders, G-mini represents a valuable animal model for the study of Alzheimer’s disease (Kragh et al., 2009; Fjord-Larsen et al., 2010) and Parkinson’s disease (Mikkelsen et al., 1999; Bjarkam et al., 2008a,b). Before G-mini can become widely applied for experimental use, it is advisable to acquire sufficient knowledge of its brain development compared with humans. To date, study of G-mini neocortical anatomy on a quantitative, histological level is limited. The aim of the present study was to expand the knowledge of the temporal pattern of G-mini postnatal neocortical neuron number development in a comprehensive series of various age-groups using neuron-specific immunohistochemistry.
Key words: Göttingen minipig, stereology, neuron number, neocortex, neurogenesis.
EXPERIMENTAL PROCEDURES
Research Laboratory for Stereology and Neuroscience, Bispebjerg University Hospital, 2400 Copenhagen NV, Denmark
Animals and tissue sampling
The Göttingen minipig (G-mini) is one of the most popular minipigs in research (Laan et al., 2010). By 6 months, G-mini is sexually mature and will reach a full-grown weight of about 40 kg, compared with a domestic pig of 200 kg or more. The popularity of G-mini as an experimental animal is attributable to its similarity to human embryogenesis, anatomy, and physiology and thus bridges the evolutionary gap between human and other commonly used laboratory animals, such as rodents and carnivore species (Lind et al., 2007). The gestational period for the G-mini is 114 –116 days, similar to the domestic pig as G-mini is a crossbreed rather than a new race in itself. However, when compared with the domestic pig, G-mini has a smaller size and is therefore easier to handle. In term of brain mass, a neonatal G-mini brain weighs about 27 g in contrast to a neonatal domestic pig brain of 29 g. In the adult period, G-mini and domestic pig brains weigh 79 and 134 g, respectively (Jelsing et al., 2006a). Within the last decades, G-mini was mainly applied to toxicology (Bode et al., 2010), diabetes (Larsen and Rolin, 2004), and visceral allo- and xenotransplantation (Kenmo-
Thirty G-minis in five groups of postnatal aged 1 day, 14 days, 30 days, 100 days, and 2 years or older (adults) of both genders were sacrificed by a procedure approved by the Danish Animal Research Inspectorate. The G-minis were anesthetized with Zolitil50® IM (tiletamine and zolazepam) (Virbac, Kolding, Denmark) and then sacrificed with a lethal dosage of pentobarbital IV (1 ml/kg body mass, 200 mg/ml) (Lundbeck, Valby, Denmark). The brains were subsequently removed in toto and immersion fixated in 4% paraformaldehyde at 20 °C for 9 days. One hemisphere was systematically and randomly selected and cut into series of 100-m-thick coronal cryostat sections. Eight to 14 sections were collected from each hemisphere in a systematic uniform random fashion, as illustrated previously (Jelsing et al., 2006a).
Immunohistochemistry The sections from all subjects were stained in the same time periods with the same batch of reagents. On day 1, the sections were rinsed in Tris-buffered saline (TBS: 0.05 M; pH 7.6); 3% H2O2 was applied for 30 min; and antigen was retrieved with 10% DakoCytomation antigen retrieval solution (pH 6) in distilled water at 95 °C for 30 min. The sections were then incubated in TBS with 1% Triton X-100 (TBS⫹T) for 3⫻10 min and treated in 10% fetal calf serum (FCS) with TBS for 1 h. Incubation in NeuN (Chemicon MAB337) was performed for 48 h at 4 °C diluted in 10% FCS/TBS solution with the titer 1:18,000.
*Corresponding author. Tel: ⫹45-35316421; fax: ⫹45-35316434. E-mail address: [email protected] (J. Hou). Abbreviations: CV, coefficient of variation; G-mini, Göttingen minipig; TBS, Tris-buffered saline; TBS⫹T, TBS with 1% Triton X-100.
0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.08.025
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J. Hou et al. / Neuroscience 195 (2011) 176 –179
177
1 1 1 ⫻ ⫻ ⫻ Q⫺⫻2, ssf asf hsf 兺 where N is the estimated total number of neurons in the neocortex, ssf is the section sampling fraction, asf is the area sampling fraction, and hsf is the height sampling fraction; Q⫺ is the total number of neurons counted, multiplied by 2 to get the bilateral neocortical number.
previously (Jelsing et al., 2006a). N⫽
Neocortical volume estimation Post-fixated neocortical volume was also estimated using the Cavalieri principle, V⫽t⫻k⫻a共p兲⫻兺P⫻2, determined by the cryostat section thickness, t⫽100 m in this study for all subjects, the inverse of sample sampling fraction, k, the area associated with each point, a(p), and the number of points superimposed on the neocortex, P. Tissue shrinkage was not measured.
Reproducibility assessment Fig. 1. High magnification micrograph of cryostat section stained with NeuN. Arrows indicate neurons. Size bar represents 20 m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
Reproducibility assessment was carried out by systematically and randomly selecting and recounting one to two sections from each brain. The re-estimated values deviated 2.7% from the original values.
On day 3, the sections were washed in TBS⫹T and incubated with 1 part Envision⫹ System-HRP (Dako K4001, Dako, Glostrup, Denmark) and 1 part TBS⫹T for 48 h at 4 °C. On day 5, the sections were washed in TBS and then developed with 9 mg DAB (Thermo Scientific #34001) per 100 ml TBS for 7 min followed by incubation in the same solution with added 20 l of 30% H2O2 for 10 min. The sections were rinsed in several shifts of TBS and PB and then mounted on Superfrost slides. Counterstaining with Cresyl Violet 0.02% (Sigma-Aldrich C5042) was performed for 15 min⫻2, dehydrated in 96% ethanol followed by 99% ethanol, cleared with xylene, and coverslipped.
Statistics
Stereology The optical fractionator technique was used to estimate the total neuron numbers in the neocortex (West, 1993). The neocortex was delineated at the white and gray matter junction. Systematically, randomly positioned grids containing counting frames were superimposed on the neocortex using the CAST-GRID software (Visiopharm, Hørsholm, Denmark) on the slides. Positively stained cells were identified as neurons; the cell bodies were used as counting items (Fig. 1). A quantitative pilot study was performed to ensure that NeuN has achieved complete tissue penetration and provided uniform signaling in the counted depth.
Population estimation The neurons were counted using a 100⫻ oil immersion lens (NA 1.40). The x-y step length and the size of the counting frame area were adjusted so that an average of 160 (range 139 –192, see Table 1) neurons was counted. The neocortical neuronal population of a subject was estimated with the formula explained in detail
The coefficient of variation (CV⫽SD/mean) was calculated. Student’s t-test was used for statistical comparison, with P⬍0.05 considered statistically significant.
RESULTS The total number of neocortical neurons in the G-mini brain was 236⫻106 (CV⫽0.051), 274⫻106 (CV⫽0.079), 264⫻106 (CV⫽0.111), 338⫻106 (CV⫽0.121), and 353⫻106 (CV⫽ 0.094) for postnatal day 1, day 14, day 30, day 100, and 2 years or older, respectively (Fig. 2, Table 2). This significant difference between the newborn and the second year (P⬍0.001) demonstrated that up to 50% of the final neocortical neurons were generated in the postnatal period. This growth in neuronal number was biphasic, with significant increase in the periods of postnatal day 1 to day 14 (P⫽0.013) and day 30 to day 100 (P⬍0.001). A temporary stabilization of neuronal number was observed from day 14 to day 30 (P⫽0.58). Additionally, from day 100 onward, the neuronal population stagnated (P⫽0.39). The hemispheric weight and neocortical volume increased almost threefold from birth to the second year or older.
DISCUSSION G-mini is regarded as a prospective model for neurological diseases because of its similarities in neural development
Table 1. Sampling parameters, mean values Age
1/ssf
⌺sect
a(frame) (m2)
Step size (m)
⌺CP
hdis (m)
tQ (m)
⌺Q
1d 14 d 30 d 100 d 2y
30 40 40 50 50
11–12 8–9 9–10 9–10 9–14
1300 1345 1263 1253 1253
3033 2750 2840 2866 2742
111 130 112 146 161
10 10 10 10 10
30.5 31.1 33.8 36.4 40.2
178 192 148 139 145
ssf, section sampling fraction; ⌺sect, number of sections; a(frame), area of the counting frame; ⌺CP, number of corner points hitting neocortex; hdis, disector height; tQ, q-weighted section thickness; ⌺Q, number of neurons counted.
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J. Hou et al. / Neuroscience 195 (2011) 176 –179
Fig. 2. Boxplots indicate mean values, points represent individual subjects, and error bars illustrate SEM. (A) The postnatal development of neocortical neurons in the G-mini. * P⫽0.013. ** P⬍0.001. (B) Neocortical volume with age in the G-mini.
to the human nervous system with regard to compositional ratio, myelination pattern, electrical activity, and in vitro cellular behavior (Dickerson and Dobbing, 1967; Flynn, 1984; Fang et al., 2005; Vodicka et al., 2005). It is also the size of the brain, the well-developed gyrification, and the ratio between cortex, white matter, and subcortical nuclei that make the G-mini a promising alternative to other experimental animals. All the above postulations were, however, derived from observations on either the cellular or gross anatomical level. It was therefore an objective of the present study to evaluate the G-mini as a potential animal model for human brain developmental and degenerative disorders on the tissue level of organization in addition to assessing its postnatal neurogenesis potential, by elucidating the temporal pattern of postnatal neocortical neuron number development in the G-mini brain. We have demonstrated the temporal pattern of postnatal development of the neocortex in terms of neuronal population, volume, and brain weight. Up to 50% of the neurons established in the matured neocortex were generated postnatally, and the neocortical volume and the hemispheric weight expanded 270% and 271%, respectively. Our data, derived from cell-specific IHC, are congruent with previous data by Jelsing et al. (2006a), who, via Giemsa staining and morphological identification of cells, have reported a postnatal increase in the number of neo-
cortical neurons at the time of birth compared with the fully grown G-mini neocortex. Furthermore, Jelsing et al. (2006b) and Guidi et al. (2011) have reported a 54% increase in the cerebellar Purkinje cells, a 60% increase in the hippocampal dentate gyrus neurons from the neonatal period to adulthood, and a large number of bromodeoxyuridine-positive cells in the subventricular zone of the lateral ventricle and hippocampal dentate gyrus postnatally, suggesting that the increase in neuron number is a global phenomenon. Such neurogenesis potential was not demonstrated in the domestic pigs (Jelsing et al., 2006a). When compared with humans, Larsen et al. (2006) have reported that the total number of neurons in the neocortex of human newborns equals the total number in adults. This finding together with several other studies suggest that neurogenesis in the human neocortex is completed at mid-gestation or at least before term, which indicates that G-mini may be an inappropriate model for the quantitative analysis of human brain development (Dobbing and Sands, 1973; Samuelsen et al., 2003). For the application of G-mini as neurodegenerative disease models, our data support the use only after postnatal day 100, when major neural organizational and developmental processes have ceased. On the other hand, an almost threefold increase in hemispheric weight and neocortical volume is consistent with previous stereological findings in the human brain, with brain weight increasing from an average of ⬇450 g in the newborn to ⬇1350 g in adult, and the neocortical volume from 195 cm3 to 489 cm3 (Pakkenberg and Gundersen, 1997; Larsen et al., 2006). Thus, in terms of the postnatal growth in brain weight and neocortical volume, the two species share similarities. The exponential growth of neocortical volume compared with the relatively slower increase in neuron numbers suggests that neuron density is an inappropriate measure of the absolute neuron number development, as it is different across age-groups and necessitates the use of stereological methods. An exceptionally low coefficient of variation is seen in the G-mini brain throughout all age-groups, which seems to be a reproducible finding and supports the continued use of G-mini in neurotoxicology studies, because perturbations may be detected with great sensitivity (Jelsing et al., 2006a,b; Nielsen et al., 2009). Notably, we revealed a period of stabilization in the neuronal number from day 14 to day 30 followed by another increase, and then stagnation after day 100. Such physiological dormancy in neural development and a secondary reactivation in the postnatal period, to the authors’ Table 2. Major estimated quantities, coefficient of variation (CV⫽ SD/mean) Age
n
Hemispheric weight (g)
Neocortical volume (cm3)
Neocortical neurons (106)
1d 14 d 30 d 100 d 2y
6 6 6 6 6
9.1 (0.055) 12.4 (0.022) 14.7 (0.073) 20.6 (0.089) 24.7 (0.117)
6.19 (0.212) 7.63 (0.095) 8.65 (0.068) 12.0 (0.155) 16.71 (0.085)
236 (0.051) 274 (0.079) 264 (0.111) 338 (0.121) 353 (0.094)
J. Hou et al. / Neuroscience 195 (2011) 176 –179
knowledge, have not been reported in other species and warrant further investigation on the growth factors and signaling pathways at play in revitalizing the dormant neuronal stem cells.
CONCLUSION Our finding demonstrated considerable postnatal (50%) increase of neuron number in the G-mini neocortex. The surge of neuron numbers took place in two time periods, from the time of birth to day 14 and from day 30 to day 100. This finding suggests that G-mini is not suitable as an animal model for the quantitative estimates of neurodevelopmental disorders and should only be used in neurodegenerative studies after day 100. The magnitude of postnatal brain growth in weight and neocortical volume was similar to human. The low CV in brain parameters supports the continued use in neurotoxicology. High postnatal proliferation potency was also demonstrated in other brain regions. In view of these features, G-mini appears to be a promising animal model to study neuroregeneration and the impact of various brain disorders on neurogenesis in the living brain. Acknowledgments—The authors are grateful to Ellegaard Göttingen Minipigs A/S (Dalmose, Denmark) for donating the minipig brains. No external funding was received for this study.
REFERENCES Bjarkam CR, Nielsen MS, Glud AN, Rosendal F, Mogensen P, Bender D, Doudet D, Møller A, Sørensen JC (2008a) Neuromodulation in a minipig MPTP model of Parkinson disease. Br J Neurosurg 22: 9 –12. Bjarkam CR, Jorgensen RL, Jensen KN, Sunde NA, Sørensen JC (2008b) Deep brain stimulation electrode anchoring using BioGlue((R)), a protective electrode covering, and a titanium microplate. J Neurosci Methods 168:151–155. Bode G, Clausing P, Gervais F, Loegsted J, Luft J, Nogues V, Sims J (2010) The utility of the minipig as an animal model in regulatory toxicology. J Pharmacol Toxicol Methods 62:196 –220. Dickerson J, Dobbing J (1967) Prenatal and postnatal growth and development of the central nervous system of the pig. Proc Biol Sci 166:384 –395. Dobbing J, Sands J (1973) Quantitative growth and development of human brain. Arch Dis Child 48:757–767. Fang M, Li J, Gong X, Antonio G, Lee F, Kwong WH, Wai SM, Yew DT (2005) Myelination of the pig’s brain: a correlated MRI and histological study. Neurosignals 14:102–108. Fjord-Larsen L, Kusk P, Tornøe J, Juliusson B, Torp M, Bjarkam CR, Nielsen MS, Handberg A, Sørensen JC, Wahlberg LU (2010) Long-term delivery of nerve growth factor by encapsulated cell
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biodelivery in the Göttingen minipig basal forebrain. Mol Ther 18:2164 –2172. Flynn TJ (1984) Developmental changes of myelin-related lipids in brain of miniature swine. Neurochem Res 9:935–945. Guidi S, Bianchi P, Olsen AK, Henningsen K, Smith DF, Bartesaghi R (2011) Postnatal neurogenesis in the hippocampal dentate gyrus and subventricular zone of the Göttingen minipig. Brain Res Bull 85:169 –179. Jelsing J, Nielsen R, Olsen AK, Grand N, Hemmingsen R, Pakkenberg B (2006a) The postnatal development of neocortical neurons and glial cells in the Göttingen minipig and the domestic pig brain. J Exp Biol 209:1454 –1462. Jelsing J, Gundersen HJ, Hielsen R, Hemmingsen R, Pakkenberg B (2006b) The postnatal development of cerebellular Purkinje cells in the Göttingen minipig estimated with a new stereological sampling technique—the Vertical bar fractionator. J Anat 209:321–331. Kenmochi T, Mullen Y, Miyamoto M, Stein E (1994) Swine as an allotransplantation model. Vet Immunol Immunopathol 43:177– 183. Kragh PM, Nielsen AL, Li J, Du Y, Lin L, Schmidt M, Bøgh IB, Holm IE, Jakobsen JE, Johansen MG, Purup S, Bolund L, Vajta G, Jørgensen AL (2009) Hemizygous minipigs produced by random gene insertion and handmade cloning express the Alzheimer’s diseasecausing dominant mutation APPsw. Transgenic Res 18:545–558. Laan JW, Brightwell J, McAnulty P, Ratky J, Stark C (2010) Regulatory acceptability of the minipig in the development of pharmaceuticals, chemicals and other products. J Pharmacol Toxicol Methods 62: 184 –195. Larsen CC, Bonde Larsen K, Bogdanovic N, Laursen H, Graem N, Samuelsen GB, Pakkenberg B (2006) Total number of cells in the human newborn telencephalic wall. Neuroscience 139:999 –1003. Larsen MO, Rolin B (2004) Use of the Göttingen minipig as a model of diabetes, with special focus on type 1 diabetes research. ILAR J 45:303–313. Lind NM, Moustgaard A, Jelsing J, Vajta G, Cumming P, Hansen AK (2007) The use of pigs in neuroscience: modeling brain disorders. Neurosci Biobehav Rev 31:728 –751. Mikkelsen M, Møller A, Jensen LH, Pedersen A, Harajehi JB, Pakkenberg H (1999) MPTP-induced Parkinsonism in minipigs: a behavioral, biochemical, and histological study. Neurotoxicol Teratol 21:169 –175. Nielsen MS, Sørensen JC, Bjarkam CR (2009) The substantia nigra pars compacta of the Göttingen minipig: an anatomical and stereological study. Brain Struct Funct 213:481– 488. Pakkenberg B, Gundersen HJ (1997) Neocortical neuron number in humans: effect of sex and age. J Comp Neurol 384:312–320. Sachs DH (1994) The pig as a potential xenograft donor. Vet Immunol Immunopathol 43:185–191. Samuelsen GB, Larsen KB, Bogdanovic N, Laursen H, Graem N, Larsen JF, Pakkenberg B (2003) The changing number of cells in the human fetal forebrain and its subdivisions: a stereological analysis. Cereb Cortex 13:115–122. Vodicka P, Smetana K, Dvoránková B, Emerick T, Xu YZ, Ourednik J, Ourednik V, Motlík J (2005) The miniature pig as an animal model in biomedical research. Ann N Y Acad Sci 1049:161–171. West MJ (1993) New stereological methods for counting neurons. Neurobiol Aging 14:275–285.
(Accepted 11 August 2011) (Available online 19 August 2011)
THE TEMPORAL PATTERN OF POSTNATAL NEUROGENESIS FOUND IN THE NEOCORTEX OF THE GÖTTINGEN MINIPIG BRAIN J. HOU,* N. ERIKSEN AND B. PAKKENBERG
Abstract—The Göttingen minipig (G-mini) is increasingly used as a non-primate model for human neurological diseases. We applied design-based stereology on five groups of G-minis aged 1 day, 14 days, 30 days, 100 days, and 2 years or older to estimate the pattern of postnatal neuron number development in the neocortex. Two time periods for the postnatal increase of neocortical neuron number were observed from the time of birth to day 14 (Pⴝ0.013) and from day 30 to day 100 (P<0.001). No significant change in neuron number was found from day 14to 30 (Pⴝ0.58) and day 100 onward (Pⴝ0.39). The average estimated total number of neurons in the neocortex was 236, 274, 264, 338, and 353 million, respectively. Since neurogenesis and neuronal migration in the human neocortex are generally accepted to be complete before term, the application of G-mini as human disease models may be inappropriate before day 100. However, G-mini may serve as a valuable model for the studies of ongoing neurogenesis in the living brain. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
chi et al., 1994; Sachs, 1994). In recent years, G-mini is winning increasing popularity in neuroscience, as it reflects the geometry and complexity of the cytoarchitecture found in the human CNS, while at the same time circumvents the technical and organizational difficulties and high costs related to non-human primate models. Recently, with the development of transgenic and chemically induced models of neurodegenerative disorders, G-mini represents a valuable animal model for the study of Alzheimer’s disease (Kragh et al., 2009; Fjord-Larsen et al., 2010) and Parkinson’s disease (Mikkelsen et al., 1999; Bjarkam et al., 2008a,b). Before G-mini can become widely applied for experimental use, it is advisable to acquire sufficient knowledge of its brain development compared with humans. To date, study of G-mini neocortical anatomy on a quantitative, histological level is limited. The aim of the present study was to expand the knowledge of the temporal pattern of G-mini postnatal neocortical neuron number development in a comprehensive series of various age-groups using neuron-specific immunohistochemistry.
Key words: Göttingen minipig, stereology, neuron number, neocortex, neurogenesis.
EXPERIMENTAL PROCEDURES
Research Laboratory for Stereology and Neuroscience, Bispebjerg University Hospital, 2400 Copenhagen NV, Denmark
Animals and tissue sampling
The Göttingen minipig (G-mini) is one of the most popular minipigs in research (Laan et al., 2010). By 6 months, G-mini is sexually mature and will reach a full-grown weight of about 40 kg, compared with a domestic pig of 200 kg or more. The popularity of G-mini as an experimental animal is attributable to its similarity to human embryogenesis, anatomy, and physiology and thus bridges the evolutionary gap between human and other commonly used laboratory animals, such as rodents and carnivore species (Lind et al., 2007). The gestational period for the G-mini is 114 –116 days, similar to the domestic pig as G-mini is a crossbreed rather than a new race in itself. However, when compared with the domestic pig, G-mini has a smaller size and is therefore easier to handle. In term of brain mass, a neonatal G-mini brain weighs about 27 g in contrast to a neonatal domestic pig brain of 29 g. In the adult period, G-mini and domestic pig brains weigh 79 and 134 g, respectively (Jelsing et al., 2006a). Within the last decades, G-mini was mainly applied to toxicology (Bode et al., 2010), diabetes (Larsen and Rolin, 2004), and visceral allo- and xenotransplantation (Kenmo-
Thirty G-minis in five groups of postnatal aged 1 day, 14 days, 30 days, 100 days, and 2 years or older (adults) of both genders were sacrificed by a procedure approved by the Danish Animal Research Inspectorate. The G-minis were anesthetized with Zolitil50® IM (tiletamine and zolazepam) (Virbac, Kolding, Denmark) and then sacrificed with a lethal dosage of pentobarbital IV (1 ml/kg body mass, 200 mg/ml) (Lundbeck, Valby, Denmark). The brains were subsequently removed in toto and immersion fixated in 4% paraformaldehyde at 20 °C for 9 days. One hemisphere was systematically and randomly selected and cut into series of 100-m-thick coronal cryostat sections. Eight to 14 sections were collected from each hemisphere in a systematic uniform random fashion, as illustrated previously (Jelsing et al., 2006a).
Immunohistochemistry The sections from all subjects were stained in the same time periods with the same batch of reagents. On day 1, the sections were rinsed in Tris-buffered saline (TBS: 0.05 M; pH 7.6); 3% H2O2 was applied for 30 min; and antigen was retrieved with 10% DakoCytomation antigen retrieval solution (pH 6) in distilled water at 95 °C for 30 min. The sections were then incubated in TBS with 1% Triton X-100 (TBS⫹T) for 3⫻10 min and treated in 10% fetal calf serum (FCS) with TBS for 1 h. Incubation in NeuN (Chemicon MAB337) was performed for 48 h at 4 °C diluted in 10% FCS/TBS solution with the titer 1:18,000.
*Corresponding author. Tel: ⫹45-35316421; fax: ⫹45-35316434. E-mail address: [email protected] (J. Hou). Abbreviations: CV, coefficient of variation; G-mini, Göttingen minipig; TBS, Tris-buffered saline; TBS⫹T, TBS with 1% Triton X-100.
0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.08.025
176
J. Hou et al. / Neuroscience 195 (2011) 176 –179
177
1 1 1 ⫻ ⫻ ⫻ Q⫺⫻2, ssf asf hsf 兺 where N is the estimated total number of neurons in the neocortex, ssf is the section sampling fraction, asf is the area sampling fraction, and hsf is the height sampling fraction; Q⫺ is the total number of neurons counted, multiplied by 2 to get the bilateral neocortical number.
previously (Jelsing et al., 2006a). N⫽
Neocortical volume estimation Post-fixated neocortical volume was also estimated using the Cavalieri principle, V⫽t⫻k⫻a共p兲⫻兺P⫻2, determined by the cryostat section thickness, t⫽100 m in this study for all subjects, the inverse of sample sampling fraction, k, the area associated with each point, a(p), and the number of points superimposed on the neocortex, P. Tissue shrinkage was not measured.
Reproducibility assessment Fig. 1. High magnification micrograph of cryostat section stained with NeuN. Arrows indicate neurons. Size bar represents 20 m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
Reproducibility assessment was carried out by systematically and randomly selecting and recounting one to two sections from each brain. The re-estimated values deviated 2.7% from the original values.
On day 3, the sections were washed in TBS⫹T and incubated with 1 part Envision⫹ System-HRP (Dako K4001, Dako, Glostrup, Denmark) and 1 part TBS⫹T for 48 h at 4 °C. On day 5, the sections were washed in TBS and then developed with 9 mg DAB (Thermo Scientific #34001) per 100 ml TBS for 7 min followed by incubation in the same solution with added 20 l of 30% H2O2 for 10 min. The sections were rinsed in several shifts of TBS and PB and then mounted on Superfrost slides. Counterstaining with Cresyl Violet 0.02% (Sigma-Aldrich C5042) was performed for 15 min⫻2, dehydrated in 96% ethanol followed by 99% ethanol, cleared with xylene, and coverslipped.
Statistics
Stereology The optical fractionator technique was used to estimate the total neuron numbers in the neocortex (West, 1993). The neocortex was delineated at the white and gray matter junction. Systematically, randomly positioned grids containing counting frames were superimposed on the neocortex using the CAST-GRID software (Visiopharm, Hørsholm, Denmark) on the slides. Positively stained cells were identified as neurons; the cell bodies were used as counting items (Fig. 1). A quantitative pilot study was performed to ensure that NeuN has achieved complete tissue penetration and provided uniform signaling in the counted depth.
Population estimation The neurons were counted using a 100⫻ oil immersion lens (NA 1.40). The x-y step length and the size of the counting frame area were adjusted so that an average of 160 (range 139 –192, see Table 1) neurons was counted. The neocortical neuronal population of a subject was estimated with the formula explained in detail
The coefficient of variation (CV⫽SD/mean) was calculated. Student’s t-test was used for statistical comparison, with P⬍0.05 considered statistically significant.
RESULTS The total number of neocortical neurons in the G-mini brain was 236⫻106 (CV⫽0.051), 274⫻106 (CV⫽0.079), 264⫻106 (CV⫽0.111), 338⫻106 (CV⫽0.121), and 353⫻106 (CV⫽ 0.094) for postnatal day 1, day 14, day 30, day 100, and 2 years or older, respectively (Fig. 2, Table 2). This significant difference between the newborn and the second year (P⬍0.001) demonstrated that up to 50% of the final neocortical neurons were generated in the postnatal period. This growth in neuronal number was biphasic, with significant increase in the periods of postnatal day 1 to day 14 (P⫽0.013) and day 30 to day 100 (P⬍0.001). A temporary stabilization of neuronal number was observed from day 14 to day 30 (P⫽0.58). Additionally, from day 100 onward, the neuronal population stagnated (P⫽0.39). The hemispheric weight and neocortical volume increased almost threefold from birth to the second year or older.
DISCUSSION G-mini is regarded as a prospective model for neurological diseases because of its similarities in neural development
Table 1. Sampling parameters, mean values Age
1/ssf
⌺sect
a(frame) (m2)
Step size (m)
⌺CP
hdis (m)
tQ (m)
⌺Q
1d 14 d 30 d 100 d 2y
30 40 40 50 50
11–12 8–9 9–10 9–10 9–14
1300 1345 1263 1253 1253
3033 2750 2840 2866 2742
111 130 112 146 161
10 10 10 10 10
30.5 31.1 33.8 36.4 40.2
178 192 148 139 145
ssf, section sampling fraction; ⌺sect, number of sections; a(frame), area of the counting frame; ⌺CP, number of corner points hitting neocortex; hdis, disector height; tQ, q-weighted section thickness; ⌺Q, number of neurons counted.
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J. Hou et al. / Neuroscience 195 (2011) 176 –179
Fig. 2. Boxplots indicate mean values, points represent individual subjects, and error bars illustrate SEM. (A) The postnatal development of neocortical neurons in the G-mini. * P⫽0.013. ** P⬍0.001. (B) Neocortical volume with age in the G-mini.
to the human nervous system with regard to compositional ratio, myelination pattern, electrical activity, and in vitro cellular behavior (Dickerson and Dobbing, 1967; Flynn, 1984; Fang et al., 2005; Vodicka et al., 2005). It is also the size of the brain, the well-developed gyrification, and the ratio between cortex, white matter, and subcortical nuclei that make the G-mini a promising alternative to other experimental animals. All the above postulations were, however, derived from observations on either the cellular or gross anatomical level. It was therefore an objective of the present study to evaluate the G-mini as a potential animal model for human brain developmental and degenerative disorders on the tissue level of organization in addition to assessing its postnatal neurogenesis potential, by elucidating the temporal pattern of postnatal neocortical neuron number development in the G-mini brain. We have demonstrated the temporal pattern of postnatal development of the neocortex in terms of neuronal population, volume, and brain weight. Up to 50% of the neurons established in the matured neocortex were generated postnatally, and the neocortical volume and the hemispheric weight expanded 270% and 271%, respectively. Our data, derived from cell-specific IHC, are congruent with previous data by Jelsing et al. (2006a), who, via Giemsa staining and morphological identification of cells, have reported a postnatal increase in the number of neo-
cortical neurons at the time of birth compared with the fully grown G-mini neocortex. Furthermore, Jelsing et al. (2006b) and Guidi et al. (2011) have reported a 54% increase in the cerebellar Purkinje cells, a 60% increase in the hippocampal dentate gyrus neurons from the neonatal period to adulthood, and a large number of bromodeoxyuridine-positive cells in the subventricular zone of the lateral ventricle and hippocampal dentate gyrus postnatally, suggesting that the increase in neuron number is a global phenomenon. Such neurogenesis potential was not demonstrated in the domestic pigs (Jelsing et al., 2006a). When compared with humans, Larsen et al. (2006) have reported that the total number of neurons in the neocortex of human newborns equals the total number in adults. This finding together with several other studies suggest that neurogenesis in the human neocortex is completed at mid-gestation or at least before term, which indicates that G-mini may be an inappropriate model for the quantitative analysis of human brain development (Dobbing and Sands, 1973; Samuelsen et al., 2003). For the application of G-mini as neurodegenerative disease models, our data support the use only after postnatal day 100, when major neural organizational and developmental processes have ceased. On the other hand, an almost threefold increase in hemispheric weight and neocortical volume is consistent with previous stereological findings in the human brain, with brain weight increasing from an average of ⬇450 g in the newborn to ⬇1350 g in adult, and the neocortical volume from 195 cm3 to 489 cm3 (Pakkenberg and Gundersen, 1997; Larsen et al., 2006). Thus, in terms of the postnatal growth in brain weight and neocortical volume, the two species share similarities. The exponential growth of neocortical volume compared with the relatively slower increase in neuron numbers suggests that neuron density is an inappropriate measure of the absolute neuron number development, as it is different across age-groups and necessitates the use of stereological methods. An exceptionally low coefficient of variation is seen in the G-mini brain throughout all age-groups, which seems to be a reproducible finding and supports the continued use of G-mini in neurotoxicology studies, because perturbations may be detected with great sensitivity (Jelsing et al., 2006a,b; Nielsen et al., 2009). Notably, we revealed a period of stabilization in the neuronal number from day 14 to day 30 followed by another increase, and then stagnation after day 100. Such physiological dormancy in neural development and a secondary reactivation in the postnatal period, to the authors’ Table 2. Major estimated quantities, coefficient of variation (CV⫽ SD/mean) Age
n
Hemispheric weight (g)
Neocortical volume (cm3)
Neocortical neurons (106)
1d 14 d 30 d 100 d 2y
6 6 6 6 6
9.1 (0.055) 12.4 (0.022) 14.7 (0.073) 20.6 (0.089) 24.7 (0.117)
6.19 (0.212) 7.63 (0.095) 8.65 (0.068) 12.0 (0.155) 16.71 (0.085)
236 (0.051) 274 (0.079) 264 (0.111) 338 (0.121) 353 (0.094)
J. Hou et al. / Neuroscience 195 (2011) 176 –179
knowledge, have not been reported in other species and warrant further investigation on the growth factors and signaling pathways at play in revitalizing the dormant neuronal stem cells.
CONCLUSION Our finding demonstrated considerable postnatal (50%) increase of neuron number in the G-mini neocortex. The surge of neuron numbers took place in two time periods, from the time of birth to day 14 and from day 30 to day 100. This finding suggests that G-mini is not suitable as an animal model for the quantitative estimates of neurodevelopmental disorders and should only be used in neurodegenerative studies after day 100. The magnitude of postnatal brain growth in weight and neocortical volume was similar to human. The low CV in brain parameters supports the continued use in neurotoxicology. High postnatal proliferation potency was also demonstrated in other brain regions. In view of these features, G-mini appears to be a promising animal model to study neuroregeneration and the impact of various brain disorders on neurogenesis in the living brain. Acknowledgments—The authors are grateful to Ellegaard Göttingen Minipigs A/S (Dalmose, Denmark) for donating the minipig brains. No external funding was received for this study.
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(Accepted 11 August 2011) (Available online 19 August 2011)