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A tissue retrieval and postharvest processing regimen for rodent reproductive tissues compatible with long-term storage on the international space station and postflight biospecimen sharing program.

Gupta V, Holets-Bondar L, Roby KF, Enders G, Tash JS - Biomed Res Int (2015)

Bottom Line: Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging.Postfixation processing was also standardized for safe shipment back to our laboratory.Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular & Integrative Physiology, University of Kansas Medical Center, Mail Stop 3050, 3901 Rainbow Boulevard, HLSIC 3098, Kansas City, KS 66160, USA.

ABSTRACT
Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging. Specimens must be harvested with minimal time after landing to minimize postflight readaptation alterations in protein expression/translation, posttranslational modifications, and expression, as well as changes in gene expression and tissue histological degradation after euthanasia. We report the development of a widely applicable strategy for determining the window of optimal species-specific and tissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs. We also determined methods for ISS-compatible long-term tissue storage (10 months at -80°C) that yield recovery of high quality mRNA and protein for western analysis after sample return. Our focus was reproductive tissues. The time following euthanasia where tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and 3 hours. RNA quality was preserved in key reproductive tissues fixed in RNAlater up to 40 min after euthanasia. Postfixation processing was also standardized for safe shipment back to our laboratory. Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

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Related in: MedlinePlus

Effect of delayed processing on C57Bl/6J testicular morphology. Each panel represents light microscopy (40x objective) of sections of adult mouse testis stained with hematoxylin and eosin (HE) (magnification bar is 100 μm). (a) Tissues kept on ice for 0 to 2.5 hr after harvesting; (b) tissues allowed to remain in the carcass for 0 to 2.5 hr; (c) tissues removed and kept at RT for 0 to 2.5 hr in Ham's F10 after harvesting.
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Related In: Results  -  Collection


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fig1: Effect of delayed processing on C57Bl/6J testicular morphology. Each panel represents light microscopy (40x objective) of sections of adult mouse testis stained with hematoxylin and eosin (HE) (magnification bar is 100 μm). (a) Tissues kept on ice for 0 to 2.5 hr after harvesting; (b) tissues allowed to remain in the carcass for 0 to 2.5 hr; (c) tissues removed and kept at RT for 0 to 2.5 hr in Ham's F10 after harvesting.

Mentions: Testes harvested any time between time 0 after euthanasia up to 2.5 hr after euthanasia showed histological properties comparable to the time 0-harvested mice. For comparative purposes, the 0 min harvested testes represent the control for subsequent time points for each storage treatment, respectively. Histological quality of the testes was excellent in all postharvest treatment procedures; namely, tissues were kept in the carcass until fixation or fixed in Bouin's immediately or stored in Hams F10 on ice or RT before fixation. Testis tubules appeared normal in all treatment groups compared to the 0 min controls, with no shrinkage of tissue and complete retention of histologic architectural details (Figures 1(a), 1(b), and 1(c)).


A tissue retrieval and postharvest processing regimen for rodent reproductive tissues compatible with long-term storage on the international space station and postflight biospecimen sharing program.

Gupta V, Holets-Bondar L, Roby KF, Enders G, Tash JS - Biomed Res Int (2015)

Effect of delayed processing on C57Bl/6J testicular morphology. Each panel represents light microscopy (40x objective) of sections of adult mouse testis stained with hematoxylin and eosin (HE) (magnification bar is 100 μm). (a) Tissues kept on ice for 0 to 2.5 hr after harvesting; (b) tissues allowed to remain in the carcass for 0 to 2.5 hr; (c) tissues removed and kept at RT for 0 to 2.5 hr in Ham's F10 after harvesting.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4309301&req=5

fig1: Effect of delayed processing on C57Bl/6J testicular morphology. Each panel represents light microscopy (40x objective) of sections of adult mouse testis stained with hematoxylin and eosin (HE) (magnification bar is 100 μm). (a) Tissues kept on ice for 0 to 2.5 hr after harvesting; (b) tissues allowed to remain in the carcass for 0 to 2.5 hr; (c) tissues removed and kept at RT for 0 to 2.5 hr in Ham's F10 after harvesting.
Mentions: Testes harvested any time between time 0 after euthanasia up to 2.5 hr after euthanasia showed histological properties comparable to the time 0-harvested mice. For comparative purposes, the 0 min harvested testes represent the control for subsequent time points for each storage treatment, respectively. Histological quality of the testes was excellent in all postharvest treatment procedures; namely, tissues were kept in the carcass until fixation or fixed in Bouin's immediately or stored in Hams F10 on ice or RT before fixation. Testis tubules appeared normal in all treatment groups compared to the 0 min controls, with no shrinkage of tissue and complete retention of histologic architectural details (Figures 1(a), 1(b), and 1(c)).

Bottom Line: Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging.Postfixation processing was also standardized for safe shipment back to our laboratory.Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular & Integrative Physiology, University of Kansas Medical Center, Mail Stop 3050, 3901 Rainbow Boulevard, HLSIC 3098, Kansas City, KS 66160, USA.

ABSTRACT
Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging. Specimens must be harvested with minimal time after landing to minimize postflight readaptation alterations in protein expression/translation, posttranslational modifications, and expression, as well as changes in gene expression and tissue histological degradation after euthanasia. We report the development of a widely applicable strategy for determining the window of optimal species-specific and tissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs. We also determined methods for ISS-compatible long-term tissue storage (10 months at -80°C) that yield recovery of high quality mRNA and protein for western analysis after sample return. Our focus was reproductive tissues. The time following euthanasia where tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and 3 hours. RNA quality was preserved in key reproductive tissues fixed in RNAlater up to 40 min after euthanasia. Postfixation processing was also standardized for safe shipment back to our laboratory. Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

Show MeSH
Related in: MedlinePlus