Identifikacija, klasifikacija i kvantifikacija razvojnih stadija postnatalnog korteksa Monodelphis domestica pomoću metode izotropne frakcionacije

One of the less understood branches of modern neurobiology is the (in)ability of adult mammals to regenerate their central nervous system (CNS) after injury. This is possible in species such as octopuses and lizards but is lacking in mammals as it becomes lost during development. Monodelphis domestica, the grey South American short‐tailed opossum is one of the few mammals that can fully and functionally regenerate their CNS after injury in the first two postnatal weeks, whereas most other species from the mammalian class lose their regenerative capacity soon after birth. Opossum are born very immature and continue to develop latched to the mother's belly, which therefore makes them excellent candidates for neuroregenerative and neurodegenerative research. Corticogenesis of the opossum brain is mostly postnatal, but in contrast to rodents has not been sufficiently explored. To better understand the process of corticogenesis and transition of heterogenous neural stem cells (NSC) into neurons, a specific method is required for precise quantification of their number and characterization of their expression profile. For this purpose, the isotropic fractionator (IFR) method has been modified and optimized for work with opossum tissues and under inverted fluorescent microscope, and used to identify, quantify, and classify cortical cell lines of the developing opossum brain. Data gained on fixed and homogenized cortical tissue samples has been compared with the recently established primary dissociated opossum cortical cell cultures, which also exhibit regenerative potential during different developing periods in vitro. Additionally, the robustness and precision of the IFR has been validated for opossum tissue by comparing the results with immunohistochemically labelled cortical tissue slices and primary dissociated opossum cortical cell cultures. Three timepoints of interest have been chosen for research of postnatal development of the opossum cortex. The timepoints have been previously examined on the opossum spinal cord, which gave us the cerebral timepoints in conjunction with the knowledge that the maturation process of developing spinal cords starts in cervical and ends in lumbar regions. The timepoints chosen are: postnatal day 5‐6 (P5‐6), where the regeneration is still possible; P17‐18, where the regenerative properties cease, but the brain is still not fully developed; and P30, where all the cortical structures are present, with a variety of cell lines. We successfully developed IFR for opossum tissues at various postnatal ages and for the inverted microscope, both as a one‐day and two‐day protocol variants. Additionally, we showed that many of the existing antibodies can be efficiently used on opossum due to the high protein homology between opossum and the immunogen/referent animal for which the antibodies were developed.Jedna od najslabije istraženih grana moderne neurobiologije je (ne)mogućnost odraslih sisavaca da regeneriraju središnji živčani sustav (SŽS) nakon ozljede. Regeneracija je moguća u nižih vrsta kao što su hobotnice i gušteri, ali je rijetka u sisavaca, te se gubi tokom razvoja. Monodelphis domestica, sivi južnoamerički kratkorepi oposum je jedan od rijetkih sisavaca koji postnatalno mogu potpuno regenerirati SŽS nakon ozljede, zadržavajući svu njegovu funkcionalnost. Takva regeneracija se može dogoditi unutar prva dva postnatalna tjedna, dok većina ostalih sisavaca gubi mogućnost regeneracija odmah nakon poroda. Oposumi se rađaju jako nerazvijeni i nastavljaju rast i razvoj pričvršćeni majci na trbuhu. Zbog tih osobina su izvrsni kandidati za istraživanja neuroregeneracie i neurodegeneracije. Kortikogeneza mozga oposuma odvija se većinom postnatalno, ali za razliku od glodavaca nije dostatno istražena. Kako bi se bolje razumio proces kortikogeneze i tranzicija heterogenih neuralnih matičnih stanica u neurone, potrebna je metoda s kojom se može dobiti precizna kvantifikacija njihovog broja i karakterizacija njihove ekspresije proteina. Metoda izotropne frakcionacije (IFR) je u tu svrhu modificirana, te je optimizirana za rad s tkivima oposuma pod invertnim fluorescentnim mikroskopom. Koristi se za identifikaciju, kvantifikaciju i klasifikaciju staničnih linija u korteksu oposuma u razvoju. Podatci dobiveni na fiksiranim i homogeniziranim uzorcima tkiva korteksa su uspoređeni sa primarnim disociranim staničnim kulturama korteksa oposuma, nedavno uspostavljenima u našem laboratoriju. One također pokazuju regenerativne sposobnosti u različitim in vitro razvojnim periodima. Robustnost i preciznost IFR metode je također validirana usporedbom rezultata sa imunohistokemijskim prerezima tkiva korteksa i primarnim disociranim staničnim kulturama korteksa oposuma. Izabrali smo tri razvojna trenutka za istraživanje postnatalnog razvoja korteksa oposuma. Ti razvojni trenutci su detektirani u istraživanjima leđne moždine oposuma in vivo i in vitro, s posebnim naglaskon na to da maturacija SŽS počinje u cervikalnoj, a završava u lumbarnoj regiji. Odabrani razvojni trenutci su postnatalni dan 5‐6 (P5‐6), kada je regeneracija SŽS još uvijek moguća; P17‐18, kada regenerativne sposobnosti prestaju, ali mozak oposuma i dalje nije razvijen do kraja; te P30, kada je prisutna definirana kortikalna struktura. Uspješno smo razvili metodu IFR za tkiva oposuma različitih starosti za invertni mikroskop, kao protokol za jedan i dva dana. Dodatno smo pokazali kako se mnoga postojeća antitijela mogu koristiti na oposumima zbog visoke homologije između proteina u oposuma i imunogene/referentne životinje za koju su antitijela napravljena

Identifikacija, klasifikacija i kvantifikacija razvojnih stadija postnatalnog korteksa Monodelphis domestica pomoću metode izotropne frakcionacije

One of the less understood branches of modern neurobiology is the (in)ability of adult mammals to regenerate their central nervous system (CNS) after injury. This is possible in species such as octopuses and lizards but is lacking in mammals as it becomes lost during development. Monodelphis domestica, the grey South American short‐tailed opossum is one of the few mammals that can fully and functionally regenerate their CNS after injury in the first two postnatal weeks, whereas most other species from the mammalian class lose their regenerative capacity soon after birth. Opossum are born very immature and continue to develop latched to the mother's belly, which therefore makes them excellent candidates for neuroregenerative and neurodegenerative research. Corticogenesis of the opossum brain is mostly postnatal, but in contrast to rodents has not been sufficiently explored. To better understand the process of corticogenesis and transition of heterogenous neural stem cells (NSC) into neurons, a specific method is required for precise quantification of their number and characterization of their expression profile. For this purpose, the isotropic fractionator (IFR) method has been modified and optimized for work with opossum tissues and under inverted fluorescent microscope, and used to identify, quantify, and classify cortical cell lines of the developing opossum brain. Data gained on fixed and homogenized cortical tissue samples has been compared with the recently established primary dissociated opossum cortical cell cultures, which also exhibit regenerative potential during different developing periods in vitro. Additionally, the robustness and precision of the IFR has been validated for opossum tissue by comparing the results with immunohistochemically labelled cortical tissue slices and primary dissociated opossum cortical cell cultures. Three timepoints of interest have been chosen for research of postnatal development of the opossum cortex. The timepoints have been previously examined on the opossum spinal cord, which gave us the cerebral timepoints in conjunction with the knowledge that the maturation process of developing spinal cords starts in cervical and ends in lumbar regions. The timepoints chosen are: postnatal day 5‐6 (P5‐6), where the regeneration is still possible; P17‐18, where the regenerative properties cease, but the brain is still not fully developed; and P30, where all the cortical structures are present, with a variety of cell lines. We successfully developed IFR for opossum tissues at various postnatal ages and for the inverted microscope, both as a one‐day and two‐day protocol variants. Additionally, we showed that many of the existing antibodies can be efficiently used on opossum due to the high protein homology between opossum and the immunogen/referent animal for which the antibodies were developed.Jedna od najslabije istraženih grana moderne neurobiologije je (ne)mogućnost odraslih sisavaca da regeneriraju središnji živčani sustav (SŽS) nakon ozljede. Regeneracija je moguća u nižih vrsta kao što su hobotnice i gušteri, ali je rijetka u sisavaca, te se gubi tokom razvoja. Monodelphis domestica, sivi južnoamerički kratkorepi oposum je jedan od rijetkih sisavaca koji postnatalno mogu potpuno regenerirati SŽS nakon ozljede, zadržavajući svu njegovu funkcionalnost. Takva regeneracija se može dogoditi unutar prva dva postnatalna tjedna, dok većina ostalih sisavaca gubi mogućnost regeneracija odmah nakon poroda. Oposumi se rađaju jako nerazvijeni i nastavljaju rast i razvoj pričvršćeni majci na trbuhu. Zbog tih osobina su izvrsni kandidati za istraživanja neuroregeneracie i neurodegeneracije. Kortikogeneza mozga oposuma odvija se većinom postnatalno, ali za razliku od glodavaca nije dostatno istražena. Kako bi se bolje razumio proces kortikogeneze i tranzicija heterogenih neuralnih matičnih stanica u neurone, potrebna je metoda s kojom se može dobiti precizna kvantifikacija njihovog broja i karakterizacija njihove ekspresije proteina. Metoda izotropne frakcionacije (IFR) je u tu svrhu modificirana, te je optimizirana za rad s tkivima oposuma pod invertnim fluorescentnim mikroskopom. Koristi se za identifikaciju, kvantifikaciju i klasifikaciju staničnih linija u korteksu oposuma u razvoju. Podatci dobiveni na fiksiranim i homogeniziranim uzorcima tkiva korteksa su uspoređeni sa primarnim disociranim staničnim kulturama korteksa oposuma, nedavno uspostavljenima u našem laboratoriju. One također pokazuju regenerativne sposobnosti u različitim in vitro razvojnim periodima. Robustnost i preciznost IFR metode je također validirana usporedbom rezultata sa imunohistokemijskim prerezima tkiva korteksa i primarnim disociranim staničnim kulturama korteksa oposuma. Izabrali smo tri razvojna trenutka za istraživanje postnatalnog razvoja korteksa oposuma. Ti razvojni trenutci su detektirani u istraživanjima leđne moždine oposuma in vivo i in vitro, s posebnim naglaskon na to da maturacija SŽS počinje u cervikalnoj, a završava u lumbarnoj regiji. Odabrani razvojni trenutci su postnatalni dan 5‐6 (P5‐6), kada je regeneracija SŽS još uvijek moguća; P17‐18, kada regenerativne sposobnosti prestaju, ali mozak oposuma i dalje nije razvijen do kraja; te P30, kada je prisutna definirana kortikalna struktura. Uspješno smo razvili metodu IFR za tkiva oposuma različitih starosti za invertni mikroskop, kao protokol za jedan i dva dana. Dodatno smo pokazali kako se mnoga postojeća antitijela mogu koristiti na oposumima zbog visoke homologije između proteina u oposuma i imunogene/referentne životinje za koju su antitijela napravljena

Identifikacija, klasifikacija i kvantifikacija razvojnih stadija postnatalnog korteksa Monodelphis domestica pomoću metode izotropne frakcionacije

One of the less understood branches of modern neurobiology is the (in)ability of adult mammals to regenerate their central nervous system (CNS) after injury. This is possible in species such as octopuses and lizards but is lacking in mammals as it becomes lost during development. Monodelphis domestica, the grey South American short‐tailed opossum is one of the few mammals that can fully and functionally regenerate their CNS after injury in the first two postnatal weeks, whereas most other species from the mammalian class lose their regenerative capacity soon after birth. Opossum are born very immature and continue to develop latched to the mother's belly, which therefore makes them excellent candidates for neuroregenerative and neurodegenerative research. Corticogenesis of the opossum brain is mostly postnatal, but in contrast to rodents has not been sufficiently explored. To better understand the process of corticogenesis and transition of heterogenous neural stem cells (NSC) into neurons, a specific method is required for precise quantification of their number and characterization of their expression profile. For this purpose, the isotropic fractionator (IFR) method has been modified and optimized for work with opossum tissues and under inverted fluorescent microscope, and used to identify, quantify, and classify cortical cell lines of the developing opossum brain. Data gained on fixed and homogenized cortical tissue samples has been compared with the recently established primary dissociated opossum cortical cell cultures, which also exhibit regenerative potential during different developing periods in vitro. Additionally, the robustness and precision of the IFR has been validated for opossum tissue by comparing the results with immunohistochemically labelled cortical tissue slices and primary dissociated opossum cortical cell cultures. Three timepoints of interest have been chosen for research of postnatal development of the opossum cortex. The timepoints have been previously examined on the opossum spinal cord, which gave us the cerebral timepoints in conjunction with the knowledge that the maturation process of developing spinal cords starts in cervical and ends in lumbar regions. The timepoints chosen are: postnatal day 5‐6 (P5‐6), where the regeneration is still possible; P17‐18, where the regenerative properties cease, but the brain is still not fully developed; and P30, where all the cortical structures are present, with a variety of cell lines. We successfully developed IFR for opossum tissues at various postnatal ages and for the inverted microscope, both as a one‐day and two‐day protocol variants. Additionally, we showed that many of the existing antibodies can be efficiently used on opossum due to the high protein homology between opossum and the immunogen/referent animal for which the antibodies were developed.Jedna od najslabije istraženih grana moderne neurobiologije je (ne)mogućnost odraslih sisavaca da regeneriraju središnji živčani sustav (SŽS) nakon ozljede. Regeneracija je moguća u nižih vrsta kao što su hobotnice i gušteri, ali je rijetka u sisavaca, te se gubi tokom razvoja. Monodelphis domestica, sivi južnoamerički kratkorepi oposum je jedan od rijetkih sisavaca koji postnatalno mogu potpuno regenerirati SŽS nakon ozljede, zadržavajući svu njegovu funkcionalnost. Takva regeneracija se može dogoditi unutar prva dva postnatalna tjedna, dok većina ostalih sisavaca gubi mogućnost regeneracija odmah nakon poroda. Oposumi se rađaju jako nerazvijeni i nastavljaju rast i razvoj pričvršćeni majci na trbuhu. Zbog tih osobina su izvrsni kandidati za istraživanja neuroregeneracie i neurodegeneracije. Kortikogeneza mozga oposuma odvija se većinom postnatalno, ali za razliku od glodavaca nije dostatno istražena. Kako bi se bolje razumio proces kortikogeneze i tranzicija heterogenih neuralnih matičnih stanica u neurone, potrebna je metoda s kojom se može dobiti precizna kvantifikacija njihovog broja i karakterizacija njihove ekspresije proteina. Metoda izotropne frakcionacije (IFR) je u tu svrhu modificirana, te je optimizirana za rad s tkivima oposuma pod invertnim fluorescentnim mikroskopom. Koristi se za identifikaciju, kvantifikaciju i klasifikaciju staničnih linija u korteksu oposuma u razvoju. Podatci dobiveni na fiksiranim i homogeniziranim uzorcima tkiva korteksa su uspoređeni sa primarnim disociranim staničnim kulturama korteksa oposuma, nedavno uspostavljenima u našem laboratoriju. One također pokazuju regenerativne sposobnosti u različitim in vitro razvojnim periodima. Robustnost i preciznost IFR metode je također validirana usporedbom rezultata sa imunohistokemijskim prerezima tkiva korteksa i primarnim disociranim staničnim kulturama korteksa oposuma. Izabrali smo tri razvojna trenutka za istraživanje postnatalnog razvoja korteksa oposuma. Ti razvojni trenutci su detektirani u istraživanjima leđne moždine oposuma in vivo i in vitro, s posebnim naglaskon na to da maturacija SŽS počinje u cervikalnoj, a završava u lumbarnoj regiji. Odabrani razvojni trenutci su postnatalni dan 5‐6 (P5‐6), kada je regeneracija SŽS još uvijek moguća; P17‐18, kada regenerativne sposobnosti prestaju, ali mozak oposuma i dalje nije razvijen do kraja; te P30, kada je prisutna definirana kortikalna struktura. Uspješno smo razvili metodu IFR za tkiva oposuma različitih starosti za invertni mikroskop, kao protokol za jedan i dva dana. Dodatno smo pokazali kako se mnoga postojeća antitijela mogu koristiti na oposumima zbog visoke homologije između proteina u oposuma i imunogene/referentne životinje za koju su antitijela napravljena

Label-Free Long-Term Methods for Live Cell Imaging of Neurons: New Opportunities

Time-lapse light microscopy combined with in vitro neuronal cultures has provided a significant contribution to the field of Developmental Neuroscience. The establishment of the neuronal polarity, i.e., formation of axons and dendrites, key structures responsible for inter-neuronal signaling, was described in 1988 by Dotti, Sullivan and Banker in a milestone paper that continues to be cited 30 years later. In the following decades, numerous fluorescently labeled tags and dyes were developed for live cell imaging, providing tremendous advancements in terms of resolution, acquisition speed and the ability to track specific cell structures. However, long-term recordings with fluorescence-based approaches remain challenging because of light-induced phototoxicity and/or interference of tags with cell physiology (e.g., perturbed cytoskeletal dynamics) resulting in compromised cell viability leading to cell death. Therefore, a label-free approach remains the most desirable method in long-term imaging of living neurons. In this paper we will focus on label-free high-resolution methods that can be successfully used over a prolonged period. We propose novel tools such as scanning ion conductance microscopy (SICM) or digital holography microscopy (DHM) that could provide new insights into live cell dynamics during neuronal development and regeneration after injury

SOX2 and SOX9 Expression in Developing Postnatal Opossum (Monodelphis domestica) Cortex

(1) Background: Central nervous system (CNS) development is characterized by dynamic changes in cell proliferation and differentiation. Key regulators of these transitions are the transcription factors such as SOX2 and SOX9. SOX2 is involved in the maintenance of progenitor cell state and neural stem cell multipotency, while SOX9, expressed in neurogenic niches, plays an important role in neuron/glia switch with predominant expression in astrocytes in the adult brain. (2) Methods: To validate SOX2 and SOX9 expression patterns in developing opossum (Monodelphis domestica) cortex, we used immunohistochemistry (IHC) and the isotropic fractionator method on fixed cortical tissue from comparable postnatal ages, as well as dissociated primary neuronal cultures. (3) Results: Neurons positive for both neuronal (TUJ1 or NeuN) and stem cell (SOX2) markers were identified, and their presence was confirmed with all methods and postnatal age groups (P4-6, P6-18, and P30) analyzed. SOX9 showed exclusive staining in non-neuronal cells, and it was coexpressed with SOX2. (4) Conclusions: The persistence of SOX2 expression in developing cortical neurons of M. domestica during the first postnatal month implies the functional role of SOX2 during neuronal differentiation and maturation, which was not previously reported in opossums