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Transplantation of Embryonic Cortical Tissue into Lesioned Adult Brain in Mice
将胚胎皮质组织移植到病变的成年小鼠脑中   

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Abstract

Transplantation of embryonic cortical tissue for repairing the damaged brain has provided a potential therapy for brain injury and diseases. The grafted tissue can successfully survive and participate in reestablishing the functional neural circuit of the host brain. Transplantation surgery can be combined with fluorescently labeled transgenic mice to evaluate the reconstruction of neuronal network (Falkner et al., 2016) and the repopulation of a subset of cortical cells. By using this approach, we have shown that infiltrating cells from host brain can restore the microglial population in the graft tissue (Wang et al., 2016). This protocol describes the detailed procedure of the transplantation surgery in mice, including establishing a lesion model in the host brain, preparing the embryonic cortical graft, and transplanting the embryonic cortical graft to adult brain.

Keywords: Transplantation(移植), Embryonic cortical tissue(胚胎皮质组织), Host(宿主), Adult brain(成年大脑), Graft (移植物)

Background

Most neurons in adult brain are post mitotic cells and are not capable of regenerating new daughter cells, this results in a limited ability of self-repairing of adult brain after suffering from brain injury or diseases. Replacing the damaged brain tissue with embryonic neural graft is one of the potential effective therapies to repair the damaged neural pathways in the adult brain (Tuszynski, 2007). Much attention has been drawn to this field of study since the 1970s (Das and Altman, 1972; Bjorklund and Stenevi, 1979) and remarkable successes have been achieved during the last three decades. These studies have shown that neurons in grafted tissue can successfully survive in host brain and develop efferent projections to reestablish synaptic connections between the host and donor neurons (Gaillard and Roger, 2000; Gaillard et al., 2004; Gaillard, 2007; Gaillard et al., 2007; Falkner et al., 2016). Electrophysiological evidence suggests that the grafted neurons develop functional connections in the host cortices of adult animals (Gaillard and Domballe, 2008; Santos-Torres et al., 2009; Jimenez-Diaz et al., 2011) and the data of behavioral tests indicate that the damaged functions can be partially restored after transplantation (Plumet et al., 1993; Riolobos et al., 2001; Gaillard et al., 2007). Our recent study suggests that there is an interactive relationship between the host brain and the transplanted tissue. The transplanted tissue provides neurons to repair the damaged circuit, and host brain can restore the microglial population in the grafted tissue (Wang et al., 2016). However, the survival and differentiation of other essential cell subsets (such as astrocyte and oligodendrocyte) and their roles and functions in the grafted tissue remain undetermined. We hope the approach we described here can be combined with other cutting-edge techniques to reveal the mechanism underlying the reconstructing process between the host brain and transplanted tissue.

Materials and Reagents

  1. Double-edge razor blade (SHANGHAI RAZOR BLADE, catalog number: 74-s , or Gillette, catalog number: PLATINUM-PLUS® )
  2. Microsurgical blade (Salvin Dental Specialties, catalog number: 6900 )
  3. Superglue (cyanoacrylate, Products of ALTECO CHEMICAL, catalog number: SG-12 )
  4. Gelfoam (Zhejiang AOKI Medical Dressing or Pfizer, catalog number: AZL0009034201 )
  5. 24-well cell culture plate (Corning, NY)
  6. 90 mm culture dish (Guangzhou Jet Bio-Filtration, catalog number: TCD010090 )
  7. Filter paper (Autoclaved)
  8. Toothpick (Autoclaved)
  9. Surgical sutures (Yangzhou Jinhuan Medical Apparatus Factory, material: silk, size: 5-0 UPS standard)
  10. 1 ml Insulin syringe (Shandong Weigao Group Medical Polymer, catalog number: B-D328404Z or BD, catalog number: 328404 )
  11. 5 ml plastic transfer pipette (Sterilized)
  12. Mice
    Note: Mice of both sexes at the age of 3-4 months are highly recommended to be used as host mice (recipient) in this protocol, and the fetus at the Embryonic day 14 (E14) or E15 (both genders) is used as donor, the strain of mice is depended on the purpose of study.
  13. 75% ethanol (Tianjin Fuyu Fine Chemical)
  14. Erythromycin ointment (paraffin based lubricant is also recommended)
  15. Iodine tincture
  16. Ketamine (Fujian Gutian Parma, catalog number: H35020148 )
  17. Xylazine (Sigma-Aldrich, catalog number: X1251-1G )
  18. Sodium chloride (NaCl) (Beichen fangzheng, Tianjin; or Sigma-Aldrich, catalog number: S5886 )
  19. Potassium chloride (KCl) (Haiguang, Tianjin; or Sigma-Aldrich, catalog number: P5405 )
  20. Potassium phosphate monobasic (KH2PO4) (The sixth chemical plant, Tianjin; or Sigma-Aldrich, catalog number: P5655 )
  21. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S9763 )
  22. Calcium chloride (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C7902 )
  23. Magnesium sulfate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 63138 )
  24. Na+-HEPES (Sigma-Aldrich, catalog number: H7006 )
  25. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5767 )
    Note: This product has been discontinued.
  26. Glucose
  27. Urethane (Sigma-Aldrich, catalog number: 94300 )
  28. Ketamine-Xylazine mixture (KX) (see Recipes)
  29. Phosphate buffered saline (PBS) (see Recipes)
  30. Hanks balanced salt solution (HBSS) (see Recipes)
  31. Urethane solution (see Recipes)

Equipment

  1. Dental drill (SEASHIN PRECISION, catalog number: STRONG 90 )
  2. Curved scissors, cutting edge: 14 mm, material: stainless steel (Fine Science Tools, catalog number: 14084-09 )
  3. Heating pad (Tme, model: JR-1/2 DC )
  4. Dissecting microscope (Olympus, model: SZ61 )
  5. Straight scissors, cutting edge: 14 mm, material: stainless steel (Fine Science Tools, catalog number: 14085-09 )
  6. Thin-tipped forceps (Fine Science Tools, model: Dumont #5 )
  7. Straight forceps (VETUS, catalog number: ST-14 )
  8. Curved forceps (Fine Science Tools, model: Dumont #5/45 )
  9. Custom-made steel plate (see Figure 1B)
  10. Compressed air (Sunto, catalog number: ST1005 )
  11. Biosafety cabinet (Jiangsu Sujing Group, model: BCM-1300A )
  12. Refrigerator

Software

  1. ImageJ software (http://rsb.info.nih.gov/ij)

Procedure

  1. Preparation of host mice (Figure 1A stage 1; Video 1)
    Note: Both sexes (male and female) can be used as recipients in this protocol. Embryonic tissue from the fetuses (regardless of their gender) at the Embryonic day 14 (E14) or E15 extracted from the pregnant mouse can be transplanted to ~3 host mice (a success transplantation means the cortical tissue from one embryonic cortex has been transplanted into the lesion cavity of the recipient), and thus ~3 host mice can be prepared.


    Figure 1. Experimental procedure of this protocol. A. Diagram showing the surgery procedure for transplantation, including preparation of host mice (stage 1), embryonic brain tissue preparation (stage 2) and transplant the embryonic cortical tissue to host brain (stage 3). At stage 1, an open-skull window was made on the host mouse and a lesion cavity was drilled in the cortex of host brain. At stage 2 embryonic brain was extracted from fetus mice. At stage 3, a piece of embryonic brain tissue was cut from the fetus brain, and grafted into the lesion cavity in adult mouse cortex. B. The host mouse was fixed to the custom-made steel plate. C. The cross-section view of the host brain after transplantation surgery. The open-skull window was covered with the bone flap and glued to the skull using Superglue.

    Video 1. Preparation of the host mice

    1. Deeply anesthetize the mice with an intraperitoneal injection (100 μl/20 g body weight) of ketamine-xylazine mixture (KX, see Recipes). The anesthetized mice are monitored until the pedal withdrawal response is lost.
    2. Sterilize all the surgical tools using 75% ethanol and lubricate the mouse’s eyes with erythromycin ointment (or paraffin based lubricant) to protect the eyes from drying during surgery.
    3. Moisten the hair on mouse head with water and shave the hair over the head with a razor blade.
    4. Disinfect the shaved scalp with iodine tincture and make an arc-shaped incision on the scalp (Figure 1A stage 1). Separate the scalp and expose the skull. Remove all fascia on the skull with a microsurgical blade.
    5. Mark the interested region on skull (-2.5 mm bregma, 2.5 mm lateral in this case) and adhere the skull to a custom-made metal frame (made by sticking two double-layered razor blades face to face to each other with Superglue. (A standard stereotactic frame can be also used for this surgery). Make sure the skull has been glued tightly with the metal frame and then fix the frame to a custom-made steel plate tightly (Figure 1B).
      Note: The metal frame should not be glued to the skull until the skull was completely dried, otherwise the metal frame will not be able to adhere to the skull tightly.
    6. Use a high-speed dental drill to thin the edge of a circular cranial area (approximately 2 mm in diameter) and clean the skull fragment with compressed air. Drill slowly and prevent the drill bur from puncturing the thinned skull.
      Note: Prevent the thinned area from being overheated by adding PBS (at room temperature) frequently.
    7. Remove the bone flap (the separated skull piece) gently with curved forceps after the skull has been thinned enough. Stop any bleeding from the exposed dura with a piece of gelfoam soaked in PBS.
    8. Clean the bone flap with PBS (see Recipes) and then place it into a 24-well culture plate filled with fresh PBS.
    9. Choose a cortical region (avoid large vessels) and create a traumatic lesion cavity (about 1 mm in diameter and 1 mm in depth) using the high-speed dental drill with a new disinfected drill bur.
    10. Clean the tissue debris in the traumatic lesion cavity and blood leaked from the lesion cavity with PBS soaked gelfoam. Detach the metal frame from the mouse skull and stop any further bleeding from the traumatic lesion cavity with gelfoam if necessary.
    11. Cover the exposed cortex with a piece of gelfoam soaked in PBS to stop bleeding of the lesion cavity. Place the mouse on a heating pad and monitor the state of anesthesia (keep the animal at the surgical level of anesthesia and inject more KX if necessary).
      Note: Protect the cortex from desiccation by adding PBS dropwise to the gelfoam frequently.

  2. Embryonic brain tissue preparation (Figure 1A stage 2; Video 2)
    Note: To maintain the viability of embryonic cortical tissue, try to finish the surgical process (extracting and transplanting) within 20 min. Cortical tissue extracted from the fetus at the Embryonic day 14 (E14) or E15 is suitable for embryonic cortical tissue transplantation.

    Video 2. Preparation of the embryonic brain tissue

    1. Sterilize all the surgical tools with 75% ethanol, including dissecting microscope, four culture dishes, straight scissors and two thin-tipped forceps, and keep them under the ultraviolet irradiation for 30 min in a biosafety cabinet.
    2. Prepare four culture dishes and pour 10 ml of 4 °C HBSS (see Recipes) into each culture dish for use.
      Note: Keep the HBSS in 4 °C refrigerator before use.
    3. Euthanize the pregnant mouse with a lethal dose of urethane (see Recipes) (about 400 μl) and sterilize with 75% ethanol. Perform an abdomen incision, and then remove the entire uterine horn and transfer it into the prepared culture dish with cold HBSS.
    4. Cut open the uterine horn and remove the amniotic sac of each embryo. Transfer the embryo into another culture dish.
    5. Cut off the fetal head, transfer the head to another culture dish with 4 °C HBSS, then peel away the scalp and skull under a dissection microscope. Remove the meninges gently with a pair of forceps to fully expose the embryonic brain. Dissociate the cortical tissue and keep it in another culture dish with cold HBSS.

  3. Transplant the embryonic cortical tissue to host brain (Figure 1A stage 3; Video 3)

    Video 3. Transplant the embryonic cortical tissue to host brain

    1. Take off the gelfoam on the brain of the host mouse and clear any remaining liquid or blood in the lesion cavity using gelfoam if necessary.
      Note: Bleeding from the exposed brain and lesion cavity should be stopped before next step.
    2. Gently pick up the embryonic cortical tissue in the HBSS and carefully cut a piece of embryonic cortical tissue (about 1 mm3) in HBSS with a pair of thin-tipped forceps. Place the tissue into the lesion cavity of the host brain and align the tissue to keep the same orientation as the host brain.
      Note: If there is any debris of cortical tissue remains outside of the lesion cavity, clear the debris carefully.
    3. Take out the separated bone flap from 24-well culture plate, and dry it with a piece of autoclaved filter paper and clean any debris on it if necessary.
      Note: Ensure to use the same piece of bone flap removed before.
    4. Hold the edge of the bone flap with forceps and cover it lightly to the exposed host brain. Keep it at original orientation and adjust the edge to make it fit well with the cranial window. Stop any bleeding with the gelfoam.
    5. Wait until the surgical region of the skull is dry. And then apply a small drop of Superglue with a toothpick to seal the seam surrounds the bone flap (Figure 1C).
      Note: Care should be taken to prevent the Superglue from infiltrating into the host brain.
    6. Wait ~3 min until the separated skull piece is glued tightly to the skull. Suture the scalp and disinfect it with iodine tincture. Put the mouse back to a separated cage and maintain the animal body temperature at 37 °C until it completely recovers from the anesthesia. The endogenous microglia undergo apoptotic death, and are lost within 36 h after transplantation.
    7. The survival and differentiation of transplanted tissue can be assessed at different time points from hours to weeks using intravital imaging or fixed tissue techniques. We observed that endogenous microglia of the grafted tissue were lost rapidly after transplantation (starting from 1 h to 36 h after transplantation), and then microglia from host brain infiltrated into the donor tissue at early stage after transplantation (from hours to 1 week). The proliferation of host-derived microglia lasted for at least one month and eventually restored the microglial population in grafted tissue. These data were reported in our recently published article (Scientific Reports 2016, 6:33080). On the other hand, the endogenous neurons of the grafted tissue survived and projected axons and dendrites to the host brain. In Figure 2, we show an example of transplanting a graft from a YFP H-line transgenic fetus to the brain of an adult wild type mouse. In this example, neurons of grafted tissue survived and differentiated in the host brain 2 months after transplantation and projected nerve fibers to the host brain (Figures 2A-2C).


      Figure 2. Fetal cortical tissue successful survived and differentiated in host brain. A. Neurons (green) in grafted tissue can survived and differentiated in host brain 2 months after transplantation and projected a multitude of axons and dendrites in the grafted tissue and the host brain. Iba-1 staining showed microglia (red) were uniformly distributed in the grafted tissue and the host brain. The white dash line shows the boundary between donor and recipient tissue. B-C. Magnified view of the yellow and white box regions in A, respectively, showing that neurons projected axons (arrow) and dendrites (arrowhead) and coexisted well with the microglia. The grafted cortical tissues were from a YFP H-line fetus, and the host was a wild-type adult mouse.

Data analysis

The development of the grafted tissue was examined by intravital two photon imaging or confocal imaging of fixed brain slices. The differentiation of the grafted tissue was analyzed by evaluating the projection of axons and dendrites of the fluorescently labeled neurons originated from the embryonic tissue. For confocal imaging, the stacks of images were acquired by using an Olympus confocal microscope (FV1000), and the z-projection of each stack of images was performed by using the Z Project function of ImageJ software (http://rsb.info.nih.gov/ij). The images of different channels were merged by using the Merge Channels function of ImageJ.

Notes

  1. To increase the success rate of the transplantation surgery, the fetuses should be extracted from the euthanatized pregnant mouse as soon as possible.
  2. During the preparation of donor tissue, the temperature of the embryonic tissue should be kept low by putting them into a culture dish with 4 °C HBSS.
  3. After the transplantation surgery, each host mouse should be fed in a separate cage to promote the recovery of the host animal. Typical survival rate of the recipient could reach to ~100% if good care has been taken. The success rate of the transplantation surgery is ~55%.
  4. A circular cover glass instead of the bone flap can be glued to the skull window for intravital two photon imaging.

Recipes

  1. Ketamine-xylazine mixture (KX)
    20 mg/ml ketamine
    2 mg/ml xylazine
    Dissolved in 0.9% NaCl
  2. Phosphate buffered saline (PBS)
    137 mM NaCl
    2.7 mM KCl
    1.4 mM KH2PO4
    8.0 mM Na2HPO4 at pH 7.4
    Sterilized and stored in refrigerator
  3. Hanks balanced salt solution (HBSS)
    137 mM NaCl
    2.5 mM CaCl2
    1.0 mM MgSO4
    5.0 mM KCl
    0.34 mM Na2HPO4
    10.0 mM Na+-HEPES
    1.0 mM NaHCO3
    20.0 mM glucose at pH 7.4
    Sterilized and stored in refrigerator
  4. Urethane solution
    1.5 g dissolved in 10 m water (ultrapure and sterilized)

Acknowledgments

This study was supported by National Natural Science Foundation of China (Nos. 81171174, 31471045) and Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP: 20130211110002). The method has been used in our recently study (Wang et al., 2016) and it is an adaptation of the methods used in previous publications (Gaillard and Roger, 2000; Gaillard et al., 2007).

References

  1. Bjorklund, A. and Stenevi, U. (1979). Reconstruction of the nigrostriatal dopamine pathway by intracerebral nigral transplants. Brain Res 177(3): 555-560.
  2. Das, G. D. and Altman, J. (1972). Studies on the transplantation of developing neural tissue in the mammalian brain. I. Transplantation of cerebellar slabs into the cerebellum of neonate rats. Brain Res 38(2): 233-249.
  3. Falkner, S., Grade, S., Dimou, L., Conzelmann, K. K., Bonhoeffer, T., Gotz, M. and Hubener, M. (2016). Transplanted embryonic neurons integrate into adult neocortical circuits. Nature 539(7628): 248-253.
  4. Gaillard, A., Prestoz, L., Dumartin, B., Cantereau, A., Morel, F., Roger, M. and Jaber, M. (2007). Reestablishment of damaged adult motor pathways by grafted embryonic cortical neurons. Nat Neurosci 10(10): 1294-1299.
  5. Gaillard, A. and Roger, M. (2000). Early commitment of embryonic neocortical cells to develop area-specific thalamic connections. Cereb Cortex 10(5): 443-453.
  6. Gaillard, F. (2007). Heterotopic, not homotopic, fetal occipital allografts in adult hosts project to visual-related extracortical targets. Restor Neurol Neurosci 25(2): 161-175.
  7. Gaillard, F. and Domballe, L. (2008). Fetal tissue allografts in the damaged adult visual cortex: physiology and connectivity. Restor Neurol Neurosci 26(4-5): 267-277.
  8. Gaillard, F., Domballe, L. and Gaillard, A. (2004). Fetal cortical allografts project massively through the adult cortex. Neuroscience 126(3): 631-637.
  9. Jimenez-Diaz, L., Nava-Mesa, M. O., Heredia, M., Riolobos, A. S., Gomez-Alvarez, M., Criado, J. M., de la Fuente, A., Yajeya, J. and Navarro-Lopez, J. D. (2011). Embryonic amygdalar transplants in adult rats with motor cortex lesions: a molecular and electrophysiological analysis. Front Neurol 2: 59.
  10. Plumet, J., Ebrahimi, A., Guitet, J. and Roger, M. (1993). Partial recovery of skilled forelimb reaching after transplantation of fetal cortical tissue in adult rats with motor cortex lesion - anatomical and functional aspects. Restor Neurol Neurosci 6(1): 9-27.
  11. Riolobos, A. S., Heredia, M., de la Fuente, J. A., Criado, J. M., Yajeya, J., Campos, J. and Santacana, M. (2001). Functional recovery of skilled forelimb use in rats obliged to use the impaired limb after grafting of the frontal cortex lesion with homotopic fetal cortex. Neurobiol Learn Mem 75(3): 274-292.
  12. Santos-Torres, J., Heredia, M., Riolobos, A. S., Jimenez-Diaz, L., Gomez-Bautista, V., de la Fuente, A., Criado, J. M., Navarro-Lopez, J. and Yajeya, J. (2009). Electrophysiological and synaptic characterization of transplanted neurons in adult rat motor cortex. J Neurotrauma 26(9): 1593-1607.
  13. Tuszynski, M. H. (2007). Rebuilding the brain: resurgence of fetal grafting. Nat Neurosci 10(10): 1229-1230.
  14. Wang, C., Tao, S., Fang, Y., Guo, J., Zhu, L. and Zhang, S. (2016). Infiltrating cells from host brain restore the microglial population in grafted cortical tissue. Sci Rep 6: 33080.

简介

移植胚胎皮质组织修复损伤的大脑已经为脑损伤和疾病提供了潜在的治疗方法。 移植组织可以成功地存活并参与重建宿主大脑的功能性神经回路。 移植手术可以与荧光标记的转基因小鼠结合,以评估神经元网络的重建(Falkner等,2016)和皮质细胞亚群的再次增殖。 通过使用这种方法,我们已经显示来自宿主大脑的浸润细胞可以恢复移植组织中的小胶质细胞群体(Wang等,2016)。 该方案描述了小鼠移植手术的详细步骤,包括在宿主脑中建立病变模型,制备胚胎皮质移植物,并将胚胎皮质移植物移植到成年大脑中。
【背景】成年大脑中的大多数神经元是后有丝分裂细胞,并且不能再生新的子细胞,这导致在患有脑损伤或疾病后自我修复成人脑的能力有限。用胚胎神经移植代替损伤的脑组织是修复成人大脑受损神经通路的潜在有效疗法之一(Tuszynski,2007)。自20世纪70年代以来,这个研究领域引起了很多关注(Das和Altman,1972; Bjorklund和Stenevi,1979年),在过去三十年中取得了显着的成功。这些研究表明,移植组织中的神经元可以成功地在宿主大脑中存活,并开发传播预测以重建宿主和供体神经元之间的突触连接(Gaillard和Roger,2000; Gaillard等,2004; Gaillard,2007; Gaillard et al ,2007; Falkner et al。,2016)。电生理学证据表明,移植的神经元在成年动物的主体皮层中发挥功能连接(Gaillard和Domballe,2008; Santos-Torres et al。,2009; Jimenez-Diaz et al。,2011),行为测试数据表明受损功能可以在移植后部分恢复(Plumet et al。,1993; Riolobos et al。,2001; Gaillard et al。,2007)。我们最近的研究表明,宿主大脑和移植组织之间存在互动关系。移植组织提供神经元修复损伤的电路,宿主大脑可以恢复移植组织中的小胶质细胞群体(Wang等,2016)。然而,其他必需细胞亚群(如星形胶质细胞和少突胶质细胞)的存活和分化及其在嫁接组织中的作用和功能尚未确定。我们希望我们在这里描述的方法可以与其他尖端技术结合,以揭示宿主大脑和移植组织之间重建过程的机制。

关键字:移植, 胚胎皮质组织, 宿主, 成年大脑, 移植物

材料和试剂

  1. 双刃刀片(上海RAZOR刀片,目录号:74-s或Gillette,目录号:PLATINUM-PLUS ®
  2. 显微外科刀片(Salvin Dental Specialties,目录号:6900)
  3. Superglue(氰基丙烯酸酯,ALTECO CHEMICAL的产品,目录号:SG-12)
  4. 吉非非诺(浙江AOKI医用敷料或辉瑞,目录号:AZL0009034201)
  5. 24孔细胞培养板(Corning,NY)
  6. 90mm培养皿(广州喷气生物过滤,目录号:TCD010090)
  7. 滤纸(高压灭菌器)
  8. 牙签(高压灭菌)
  9. 手术缝线(扬州金环医疗器械厂,材质:丝,尺寸:5-0 UPS标准)
  10. 1ml胰岛素注射器(山东威高集团医用聚合物,目录号:B-D328404Z或BD,目录号:328404)
  11. 5 ml塑料移液器(灭菌)
  12. 小鼠
    注意:强烈推荐使用3-4岁月龄的小鼠作为本方案的宿主小鼠(受体),胎儿胚胎第14天(E14)或E15(两性)用作供体,小鼠的菌株取决于研究的目的。
  13. 75%乙醇(天津富宇精细化工)
  14. 红霉素软膏(也推荐使用石蜡基润滑剂)
  15. 碘酊
  16. 氯胺酮(Fujian Gutian Parma,目录号:H35020148)
  17. 赛拉嗪(Sigma-Aldrich,目录号:X1251-1G)
  18. 氯化钠(NaCl)(北辰芳正,天津;或Sigma-Aldrich,目录号:S5886)
  19. 氯化钾(KCl)(天津海光;或Sigma-Aldrich,目录号:P5405)
  20. 磷酸二氢钾(KH 2 PO 4)(第六化工厂,天津;或Sigma-Aldrich,目录号:P5655)
  21. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S9763)
  22. 氯化钙(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C7902)
  23. 硫酸镁(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:63138)
  24. (Sigma-Aldrich,目录号:H7006)
  25. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S5767)
    注意:本产品已停产。
  26. 葡萄糖
  27. 聚氨酯(Sigma-Aldrich,目录号:94300)
  28. 氯胺酮 - 赛拉嗪混合物(KX)(见食谱)
  29. 磷酸盐缓冲盐水(PBS)(见食谱)
  30. 汉克斯平衡盐溶液(HBSS)(见食谱)
  31. 聚氨酯溶液(参见食谱)

设备

  1. 牙科钻(SEASHIN PRECISION,目录号:STRONG 90)
  2. 弯曲剪刀,切割刃:14毫米,材质:不锈钢(精细科学工具,目录号:14084-09)
  3. 加热垫(Tme,型号:JR-1/2 DC)
  4. 解剖显微镜(Olympus,型号:SZ61)
  5. 直剪刀,切边:14毫米,材质:不锈钢(精细科学工具,目录号:14085-09)
  6. 薄型镊子(精细科学工具,型号:Dumont#5)
  7. 直镊子(VETUS,目录号:ST-14)
  8. 弯曲镊子(精细科学工具,型号:Dumont#5/45)
  9. 定制钢板(见图1B)
  10. 压缩空气(Sunto,目录号:ST1005)
  11. 生物安全柜(江苏苏晶集团,型号:BCM-1300A)
  12. 冰箱

软件

  1. ImageJ软件( http://rsb.info.nih.gov/ij

程序

  1. 宿主小鼠的制备(图1A阶段1;视频1)
    注意:男性和女性都可以在本协议中作为收件人使用。从怀孕小鼠中提取的胚胎第14天(E14)或E15,胎儿胚胎组织(不论其性别如何)可以移植到〜3个宿主小鼠(成功移植意味着从一个胚胎皮层的皮层组织已被移植到受体的病变腔),因此可以制备〜3宿主小鼠。


    图1.该方案的实验程序 A.显示移植手术程序的图,包括宿主小鼠(第1阶段)的制备,胚胎脑组织制备(阶段2)和移植胚胎皮层组织主持大脑(第3阶段)。在第1阶段,对宿主小鼠进行开放式颅骨窗口,并在宿主脑皮质中钻孔。在第2阶段,从胎儿小鼠中提取胚胎脑。在第3阶段,从胎儿脑切割一块胚胎脑组织,并将其移植到成年小鼠皮层的损伤腔中。 B.将主机鼠固定在定制钢板上。 C.移植手术后宿主大脑的剖面图。颅骨窗口被骨瓣覆盖,并使用Superglue胶合到头骨。

    Video 1. Preparation of the host mice

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    1. 用氯胺酮 - 赛拉嗪混合物(KX,参见食谱)腹膜内注射(100μl/20g体重)深度麻醉小鼠。麻醉的小鼠被监测,直到踏板取出反应丧失
    2. 使用75%乙醇灭菌所有外科手术工具,并用红霉素软膏(或石蜡基润滑剂)润滑鼠标的眼睛,以保护手术期间的眼睛免受干燥。
    3. 用水润湿头发上的头发,用剃须刀刮头部头发。
    4. 用碘酊消毒剃光头皮,并在头皮上形成弧形切口(图1A级1)。分开头皮并暴露头骨。用显微外科刀片去除颅骨上的所有筋膜。
    5. 标记颅骨上的感兴趣区域(在这种情况下为2.5毫米宽,2.5毫米外侧),并将颅骨粘贴到定制的金属框架上(通过用Superglue将两个双层剃须刀刀片彼此面对地相连制成((一个标准的立体定位框架也可以用于这种手术),确保头骨已经与金属框架紧密地胶合,然后将框架紧紧地固定在定制的钢板上(图1B)。
      注意:金属框架不应该粘在骷髅头上,直到头骨被完全干燥,否则金属框架将无法紧贴颅骨。
    6. 使用高速牙钻使圆形颅面(直径约2 mm)的边缘变薄,并用压缩空气清洁颅骨碎片。慢慢地钻,并防止钻头刺穿稀疏的头骨。
      注意:通过频繁添加PBS(室温)防止变薄区域过热。
    7. 颅骨已经变薄后,用弯曲的镊子轻轻取出骨瓣(分离的颅骨片)。用一块浸泡在PBS中的一块凝胶凝胶阻止暴露的硬脑膜出血。
    8. 用PBS清洁骨瓣(参见食谱),然后将其放入装有新鲜PBS的24孔培养板中。
    9. 选择皮质区域(避免大血管),并使用高速牙钻和新的消毒钻孔头创建创伤性病变腔(直径约1毫米,深度1毫米)。
    10. 用PBS浸泡的海洛因清洗创伤性病变腔内的组织碎片和从损伤腔泄漏的血液。将金属框架从小鼠颅骨上取下,如有必要,可以使用海洛因从创伤性病变腔中进一步出血。
    11. 用一块浸泡在PBS中的一片海藻盖住暴露的皮层,以阻止病变腔的出血。将鼠标放在加热垫上并监测麻醉状态(使动物处于手术麻醉水平,如果需要,可以注射更多的KX)。
      注意:经常将PBS逐滴加入到海洛因中,保护皮质免受干燥。

  2. 胚胎脑组织准备(图1A阶段2;视频2)
    注意:为了保持胚胎皮质组织的活力,请尝试在20分钟内完成手术过程(提取和移植)。在胚胎第14天(E14)或E15从胎儿提取的皮层组织适用于胚胎皮层组织移植。

    Video 2. Preparation of the embryonic brain tissue

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    1. 用75%乙醇灭菌所有手术工具,包括解剖显微镜,四个培养皿,直剪刀和两个薄镊子,并在生物安全柜内将其保持在紫外线照射下30分钟。
    2. 准备四种培养皿,并将10ml 4℃HBSS(参见食谱)倒入每个培养皿中使用。
      注意:使用前请将HBSS放在4°C冰箱中。
    3. 用致死剂量的氨基酸(参见食谱)(约400μl)对怀孕的小鼠进行安乐死,并用75%乙醇灭菌。执行腹部切口,然后取出整个子宫角,并将其转移到用冷HBSS制备的培养皿中。
    4. 切开子宫角,取出各胚胎的羊膜。将胚胎转移到另一种培养皿中
    5. 切断胎头,用4°C HBSS将头部转移到另一个培养皿中,然后在夹层显微镜下剥离头皮和头骨。用一对镊子轻轻取出脑膜,以充分暴露胚胎大脑。分离皮质组织,并保持在另一种具有冷HBSS的培养皿中。

  3. 将胚胎皮质组织移植到宿主大脑(图1A阶段3;视频3)

    Video 3. Transplant the embryonic cortical tissue to host brain

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    1. 在主人小鼠的脑部脱掉海洛因,如有必要,使用海洛因清除病变腔内剩余的液体或血液。
      注意:暴露的脑部和病变腔出血应在下一步骤前停止。
    2. 轻轻取出HBSS中的胚胎皮质组织,并用一对薄薄的镊子在HBSS中小心地切割一块胚胎皮质组织(约1毫米<3>)。将组织置于宿主大脑的病变腔内,使组织保持与宿主脑相同的方向。
      注意:如果有皮质组织残留物残留在病变腔外,请仔细清除碎屑。
    3. 从24孔培养板中取出分离的骨瓣,并用一块高压灭菌的滤纸将其干燥,并在必要时清洁其上的任何碎屑。
      注意:确保使用与之前拆下的同一片骨瓣。
    4. 用镊子握住骨瓣的边缘,轻轻盖住暴露的宿主脑。保持原来的方向,调整边缘,使其适合颅窗。用格列吡芬阻止任何出血。
    5. 等到头骨的手术区域干燥。然后用牙签涂一小片Superglue,以密封围绕骨瓣的缝(图1C)。
      注意:请注意防止Superglue渗入宿主大脑。
    6. 等待〜3分钟,直到分离的颅骨片紧紧地粘在颅骨上。缝合头皮,用碘酊消毒。将鼠标放回分离的笼子,并将动物体温保持在37°C,直到完全从麻醉中恢复。内源性小神经胶质细胞凋亡死亡,移植后36小时内出现细胞凋亡。
    7. 移植组织的存活和分化可以使用活体成像或固定组织技术在不同的时间点从几小时到数周进行评估。我们观察到移植后移植组织内源性小胶质细胞(移植后1 h至36 h)迅速消失,移植后早期(从小时至1周),宿主脑小胶质细胞浸润到供体组织。宿主衍生的小胶质细胞的增殖持续至少一个月,最终恢复移植组织中的小胶质细胞群体。这些数据在我们最近发表的文章( Scientific Reports 2016,6,33080)中报道。另一方面,移植组织的内源性神经元存活并将轴突和树突投射到宿主脑。在图2中,我们示出了将移植物从YFP H线转基因胎儿移植到成年野生型小鼠的脑中的例子。在这个例子中,嫁接组织的神经元在移植后2个月存活和分化,并将神经纤维投射到宿主脑(图2A-2C)。


      图2.胎儿皮层组织在宿主大脑中成功存活并分化。 A.移植组织中的神经元(绿色)可以在移植后2个月内在宿主脑中存活并分化,并且将大量的轴突和树突投射在嫁接组织和宿主脑。 Iba-1染色显示小胶质细胞(红色)均匀分布在移植组织和宿主脑中。白色虚线表示供体和受体组织之间的边界。公元前。分别在A中的黄色和白色盒区域的放大图,显示神经元突出的轴突(箭头)和树突(箭头)并且与小胶质细胞共存。移植的皮质组织来自YFP H线胎儿,宿主是野生型成年小鼠。

数据分析

通过固定脑切片的活体两光子成像或共焦成像检查移植组织的发育。通过评估来自胚胎组织的荧光标记的神经元的轴突和树突的投影来分析移植组织的分化。对于共聚焦成像,通过使用奥林巴斯共聚焦显微镜(FV1000)获得图像堆叠,并且通过使用ImageJ软件的Z Project功能( http://rsb.info.nih.gov/ij )。通过使用ImageJ的合并频道功能合并不同频道的图像。

笔记

  1. 为了提高移植手术的成功率,胎儿应尽快从安乐死的怀孕小鼠中提取。
  2. 在供体组织的制备过程中,通过将胚胎组织放入含有4℃HBSS的培养皿中来保持较低的温度。
  3. 移植手术后,每只宿主小鼠应在单独的笼中喂养以促进宿主动物的恢复。如果采取良好的照顾,接受者的典型生存率可能达到〜100%。移植手术的成功率为〜55%
  4. 圆形盖玻璃代替骨瓣可以胶合到颅骨窗口进行活体两光子成像。

食谱

  1. 氯胺酮 - 赛拉嗪混合物(KX)
    20mg/ml氯胺酮
    2 mg/ml甲苯噻嗪
    溶于0.9%NaCl
  2. 磷酸盐缓冲盐水(PBS)
    137 mM NaCl
    2.7 mM KCl
    1.4mM KH 2 PO 4
    8.0mM Na 2 HPO 4在pH 7.4下进行 灭菌并储存在冰箱中
  3. 汉克斯平衡盐溶液(HBSS)
    137 mM NaCl
    2.5mM CaCl 2
    1.0mM MgSO 4
    5.0mM KCl
    0.34mM Na 2 HPO 4
    10.0mM Na +//- HEPES
    1.0mM NaHCO 3
    20.0 mM葡萄糖,pH 7.4 灭菌并储存在冰箱中
  4. 聚氨酯溶液
    1.5克溶于10毫升水(超纯灭菌)

致谢

本研究得到国家自然科学基金(81171174,341471045)和高等教育博士生专项研究基金(SRFDP:20130211110002)的支持。该方法已经在我们最近的研究中使用(Wang等人,2016),并且它是对以前出版物中使用的方法的改编(Gaillard和Roger,2000; Gaillard等人,/em>。,2007)。

参考

  1. Bjorklund,A.和Stenevi,U.(1979)。脑组织177(3):555-560。
  2. Das,GD和Altman,J.(1972)。  研究哺乳动物脑中发育神经组织的移植。 I.将小脑板移植到新生大鼠的小脑中。脑电图38(2):233-249。
  3. Falkner,S.,Grade,S.,Dimou,L.,Conzelmann,KK,Bonhoeffer,T.,Gotz,M.and Hubener,M。(2016)。  移植的胚胎神经元整合入成年新皮质电路。自然 539(7628) :248-253。
  4. Gaillard,A.,Prestoz,L.,Dumartin,B.,Cantereau,A.,Morel,F.,Roger,M。和Jaber,M。(2007)。通过移植胚胎皮层神经元重建损伤的成人运动途径 Nat Neurosci 10(10):1294-1299。
  5. Gaillard,A.和Roger,M。(2000)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/10847594"target ="_ blank"胚胎新皮质细胞早期致力于开发区域特异性丘脑连接。 Cereb Cortex 10(5):443-453。
  6. Gaillard,F.(2007)。异位,不是同位素,成年患者的胎枕同种异体移植物投射到视觉相关的皮质内靶标。 恢复Neurol Neurosci 25(2):161-175。
  7. Gaillard,F。和Domballe,L.(2008)。  损伤的成人视皮质中的胎儿组织同种异体移植物:生理和连通性。 Restor Neurol Neurosci 26(4-5):267-277。
  8. Gaillard,F.,Domballe,L.和Gaillard,A。(2004)。胎儿皮质同种异体移植物质大量通过成年皮质进行项目。神经科学 126(3):631-637。
  9. Jimenez-Diaz,L.,Nava-Mesa,MO,Heredia,M.,Riolobos,AS,Gomez-Alvarez,M.,Criado,JM,de la Fuente,A.,Yajeya,J.and Navarro-Lopez,JD (2011)。成年大鼠运动中的胚胎杏仁核移植皮层损伤:分子和电生理分析。前线Neurol 2:59.
  10. Plumet,J.,Ebrahimi,A.,Guitet,J.and Roger,M。(1993)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/21551727"target ="_ blank">在运动皮质损伤的成年大鼠移植胎儿皮质组织之后到达的技术前沿部分恢复 - 解剖和功能方面 Restor Neurol Neurosci 6(1):9-27。
  11. Riolobos,AS,Heredia,M.,de la Fuente,JA,Criado,JM,Yajeya,J.,Campos,J.and Santacana,M。(2001)。功能恢复老年人的技术前肢使用必须在用同侧胎儿皮质移植额叶皮层病变后使用受损肢体。 Neurobiol Learn Mem 75(3):274-292。
  12. Santos-Torres,J.,Heredia,M.,Riolobos,AS,Jimenez-Diaz,L.,Gomez-Bautista,V.,de la Fuente,A.,Criado,JM,Navarro-Lopez,J.and Yajeya, J.(2009)。移植的电生理和突触表征成年大鼠运动皮质中的神经元。 J Neurotrauma 26(9):1593-1607。
  13. Tuszynski,MH(2007)。  重建大脑:复兴的胎儿嫁接。 Nat Neurosci 10(10):1229-1230。
  14. Wang,C.,Tao,S.,Fang,Y.,Guo,J.,Zhu,L。和Zhang,S。(2016)。来自宿主脑的浸润细胞恢复移植的皮质组织中的小胶质细胞群体。 33080.
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, C., Gao, H. and Zhang, S. (2017). Transplantation of Embryonic Cortical Tissue into Lesioned Adult Brain in Mice. Bio-protocol 7(12): e2360. DOI: 10.21769/BioProtoc.2360.
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