This work was supported by the Ronald Peter Griggs Memorial Fellowship in ALS Research (to KP) and a Neuromuscular Research Partnership Program grant from the CIHR, the ALS Society of Canada, and Muscular Dystrophy Canada (JNM-69682) to CK and FMR. Additional support was also provided by the ALS Society of America (57969). We would like to thank Dr. R. Keith Humphries from the Terry Fox Laboratory/Department of Pathology, BC Cancer Agency, Vancouver, Canada and the staff at the Simon Fraser University Animal Care Unit.

NOTA: Etica Dichiarazione: Questo protocollo è stato esaminato e approvato dal Comitato di Simon Fraser University Animal Care (UACC; consentire numeri 1037K-12 e 1060K-03) ed è in conformità con il Consiglio canadese cura degli animali, la Guida NIH per la cura e l&#39;uso di animali da laboratorio, e la direttiva del Consiglio CEE. 1. Considerazioni particolari NOTA: Busulfan è citotossico. Manipolare e smaltire secondo la scheda dati di sicurezza e le linee guida istituzionali. <ol><li> Eseguire tutte le tecniche asetticamente in una cappa a flusso laminare. </li><li> Sterilizzare strumenti prima dell&#39;uso avvolgendo in un materiale di imballaggio appropriato, ad esempio un pacchetto buccia e autoclave. </li><li> Mentre il rischio di infezione è più basso con condizionata busulfan rispetto a irraggiamento, gestire gli animali solo in una cappa a flusso laminare per le prime 2 settimane dopo il trapianto. </li></ol> 2. CondizionamentoTopi riceventi <ol><li> Diluire busulfan a 3 mg / ml con soluzione salina sterile tamponata fosfato (PBS). NOTA: E &#39;importante fare una nuova diluizione del busulfan appena prima di utilizzare ogni giorno di iniezione. </li><li> Somministrare 20 mg / kg di busulfan diluito per topi riceventi tramite iniezione IP quotidiana. </li><li> Ripetere iniezioni IP giornaliera di 20 mg / kg fino busulfan una dose totale di 60-100 mg / kg è stata consegnata (cioè, per una dose totale di 80 mg / kg, somministrare 20 mg / kg di busulfan per 4 giorni consecutivi). </li></ol> 3. Isolamento del donatore di midollo osseo Cells NOTA: Questo protocollo è stato utilizzato con successo per isolare e preparare cellule BM da un massimo di 5 topi donatori. Tipicamente la resa delle cellule per il mouse è di circa 30-40.000.000 BMDCs, che è sufficiente a trapiantare 12-16 topi riceventi. Se sono necessarie più topi donatori il protocollo può avere bisogno di essere adeguato di conseguenza. <ol><li> Dopo l&#39;ultimo giorno di busulfan condizionata euthanize un topo GFP donatore (1-6 mesi) con CO 2 (o da altra procedura eutanasia accettato istituto). Per evitare complicazioni innesto utilizzano donatori singenici che sono dello stesso sesso come destinatari. </li><li> Spray mouse con etanolo al 70%. Pelle Sollevare le forbici chirurgiche addome e con fare un&#39;incisione attraverso la pelle dalla cavità addominale la gamba verso la caviglia. Afferrando il supporto, saldamente tirare la pelle dalla caviglia verso l&#39;anca esposizione del tessuto gamba. </li><li> Tagliare via muscoli e tessuto adiposo dal femore per esporre l&#39;articolazione dell&#39;anca. </li><li> Mentre delicatamente tirando sul piede di estendere la gamba, premere i forbici contro l&#39;anca. Tagliare appena sopra la testa del femore facendo attenzione a non tagliare femore stesso. </li><li> Per aiutare a mantenere la sterilità, tenere la gamba dal piede e pulire qualsiasi restante tessuto dalle ossa strofinando la superficie ossea con i tessuti in autoclave. </li><li> Separare femore e tibia tagliando attraverso l&#39;articolazione del ginocchio ecollocare il femore in un piatto di coltura contenente PBS sterile. Incubare in ghiaccio. </li><li> Rimuovere e gettare perone tagliando nei punti in cui la fibula collega alla tibia. Posizionare la tibia nel piatto cultura con il femore e incubare su ghiaccio. </li><li> Ripetere i passaggi 3,2-3,7 per l&#39;altra gamba, e se necessario, topi donatori supplementari. Dopo la rimozione delle ossa, sterilizzare gli strumenti chirurgici con un tallone sterilizzatrice a caldo o utilizzare una nuova serie di strumenti sterili per le fasi successive. </li><li> Per i femori, tenere il femore con una pinza e con le forbici chirurgiche attentamente &#39;barba&#39; distale finisce l&#39;osso. Rimuovere il meno dell&#39;osso se necessario per esporre la cavità BM. </li><li> Riempire una siringa con 3 ml di PBS sterile e inserire un ago G 23. Portava con attenzione l&#39;ago nella cavità BM e lavare la BM in un piatto di coltura sterile. Assicurati di raschiare cavità midollare con la punta dell&#39;ago per garantire la rimozione di tutte le cellule desiderate. Dopo l&#39;estrazione,assicurare che il rosso BM non è più visibile e l&#39;osso appare ora bianco. </li><li> Ripetere i passaggi 3,9-3,10 per femori successivi, mettendo in comune tutte le BM nello stesso piatto cultura. </li><li> Per la tibie, tenere la tibia con una pinza e con attenzione &#39;barba&#39; alla fine dove la tibia è stata allegata al ginocchio per esporre la cavità BM. Fare un secondo taglio lungo l&#39;osso in cui termina la BM rosso visibile. </li><li> Riempire una siringa con 3 ml di PBS sterile e inserire un ago G 25. Inserire delicatamente l&#39;ago nella cavità BM (dalla fine che era attaccato al ginocchio) e lavare la BM nel piatto di coltura contenente la BM dal femore. Raschiare le pareti della cavità BM con l&#39;ago per rimuovere tutte le cellule. Dopo l&#39;estrazione, assicurarsi che il rosso BM non è più visibile e l&#39;osso ora appare bianco. </li><li> Ripetere i passaggi 3,12-3,13 per la successiva tibie, riunendo tutti i BM nello stesso piatto cultura. </li><li> Triturare delicatamente la BM con un 1 ml pipetta punta a dissociare la cells. </li><li> Passare la sospensione cellulare attraverso un filtro a cestello 40 mM in una sterile 50 mL provetta da centrifuga. Sciacquare il piatto con PBS per ottenere tutte le cellule residue e trasferire questo attraverso il filtro nel tubo da centrifuga. </li><li> Centrifugare per 5 minuti a 450 xga 4 ° C. Rimuovere e scartare il surnatante. </li><li> Risospendere il pellet in 3 ml di eritrociti tampone di lisi. Incubare in ghiaccio per 8.5 min. </li><li> Aggiungi ~ 30 ml di PBS sterile per placare il buffer di lisi. </li><li> Centrifugare per 5 minuti a 450 xga 4 ° C. Rimuovere e scartare il surnatante. </li><li> Risospendere il pellet in 1 ml di PBS sterile. Mantenere su ghiaccio. </li><li> Prendere una piccola aliquota della omogenato cellulare e diluire con PBS (1: 100 per 1 mouse, 1: 200 per 2 topi, etc.) per quantificare il numero di cellule utilizzando un emocitometro. </li><li> Diluire la sospensione cellulare a 8 x 10 6 cellule / ml e incubare in ghiaccio fino iniezione. </li></ol> 4. trapianto di midollo osseo <ol><li> Fill una siringa da 0,5 ml (con insulina ago 28 G) con 300 ml di sospensione cellulare diluita per ogni mouse da iniettare. Assicurarsi di rimuovere eventuali bolle d&#39;aria presenti nella siringa. </li><li> Posizionare la gabbia del mouse che contiene i topi riceventi condizionati su una piastra elettrica e consentire le vene coda di dilatarsi. </li><li> Prendete un mouse destinatario e posto nel dispositivo di ritenuta. Pulire la coda con una garza chirurgica imbevuta di etanolo al 70%. </li><li> Tenere saldamente la coda e lo smusso up, inserire delicatamente l&#39;ago in una delle vene coda laterali e iniettare la sospensione cellulare. </li><li> Tenere garza chirurgica sul sito di iniezione fino a emorragia si ferma prima di introdurre il mouse in una nuova gabbia pulita. </li></ol> 5. valutare l&#39;estensione di BM chimerismo NOTA: i livelli di chimerismo sono in genere variabile nel sangue periferico dopo 1 settimana post-BMT, e come tali livelli chimerismo sono normalmente stimate almeno 2 settimane dopo BMT. Livelli di chimerism dovrebbe raggiungere&gt; 80% GFP + cellule del sangue periferico entro 3-4 settimane dopo BMT. <ol><li> Preparare tampone FACS (2 mM EDTA + 2% di siero fetale bovino in PBS). NOTA: tampone FACS può essere conservato a 4 ° C per diverse settimane. </li><li> Posizionare il mouse in un tubo di contenimento e accorciare il pelo sulla gamba posteriore, per esporre la vena safena laterale. </li><li> Cappotto sottilmente la gamba con vaselina e forare la vena safena con un ago 25 G. </li><li> Raccogliere circa 50 ml di sangue con un tubo capillare eparina rivestito. Aggiungere il sangue in una provetta micro-centrifuga contenente 1 ml di tampone FACS. Mescolare il tubo per inversione e memorizzare su ghiaccio. </li><li> Tenere garza chirurgica sul sito di iniezione fino a quando il sanguinamento si arresta prima di tornare mouse avanti alla gabbia. </li><li> Centrifugare i campioni di sangue per 5 min a 900 x g. Rimuovere e scartare il surnatante. </li><li> Risospendere il pellet in 500 ml di eritrociti buffer di lisi. Incubare in ghiaccio per 8.5 min. </li><li> Aggiungere 1 ml di tampone FACS per placare il buffer di lisi. </li><li> Centrifugare i campioni per 5 min a 900 x g. Rimuovere e scartare il surnatante. </li><li> Eseguire una seconda fase di lisi in 500 ml di buffer di lisi degli eritrociti. Incubare in ghiaccio per 8.5 min. </li><li> Aggiungere 1 ml di tampone FACS per placare il buffer di lisi. </li><li> Centrifugare i campioni per 5 min a 900 x g. Rimuovere e scartare il surnatante. Controllare che il pellet è ora bianca. NOTA: Se il pellet appare ancora rosso, un passo lisi supplementare può essere necessario prima di procedere alla fase successiva. </li><li> Risospendere il pellet in 1 ml di tampone FACS. Centrifugare i campioni per 5 min a 900 x g. Rimuovere e scartare il surnatante. </li><li> Risospendere il pellet in 300 ml di tampone FACS e quantificare + cellule GFP mediante citometria a flusso (NOTA: A questo punto qualsiasi immunocolorazione desiderato delle cellule può essere eseguita, utilizzando tecniche standard, prima analisi citofluorimetrica). </li></ol>

<ol>
	<li>Ginhoux, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+Mapping+Analysis+Reveals+That+Adult+Microglia+Derive+from+Primitive+Macrophages.">Fate Mapping Analysis Reveals That Adult Microglia Derive from Primitive Macrophages.</a> Science. 330 (6005), 841-845 (2010).</li><li>Hickey, W. F., Kimura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+microglial+cells+of+the+CNS+are+bone+marrow-derived+and+present+antigen+in+vivo.">Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo.</a> Science (New York, N. Y.). 239 (4837), 290-292 (1988).</li><li>Duran-Struuck, R., Dysko, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+bone+marrow+transplantation+(BMT):+providing+optimal+veterinary+and+husbandry+care+to+irradiated+mice+in+BMT+studies.">Principles of bone marrow transplantation (BMT): providing optimal veterinary and husbandry care to irradiated mice in BMT studies.</a> Journal of the American Association for Laboratory Animal Science: JAALAS. 48 (1), 11-22 (2009).</li><li>Jimenez, M., Ercilla, G., Martinez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+reconstitution+after+allogeneic+stem+cell+transplantation+with+reduced-intensity+conditioning+regimens.">Immune reconstitution after allogeneic stem cell transplantation with reduced-intensity conditioning regimens.</a> Leukemia. 21 (8), 1628-1637 (2007).</li><li>Ellen Nath, C., John Shaw, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Busulphan+in+blood+and+marrow+transplantation:+dose,+route,+frequency+and+role+of+therapeutic+drug+monitoring.">Busulphan in blood and marrow transplantation: dose, route, frequency and role of therapeutic drug monitoring.</a> Current clinical pharmacology. 2 (1), 75-91 (2007).</li><li>Galaup, A., Paci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+of+dimethanesulfonate+alkylating+agents:+busulfan+and+treosulfan.">Pharmacology of dimethanesulfonate alkylating agents: busulfan and treosulfan.</a> Expert Opinion on Drug Metabolism &amp; Toxicology. 9 (3), 333-347 (2013).</li><li>Lewis, C. -. A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelosuppressive+Conditioning+Using+Busulfan+Enables+Bone+Marrow+Cell+Accumulation+in+the+Spinal+Cord+of+a+Mouse+Model+of+Amyotrophic+Lateral+Sclerosis.">Myelosuppressive Conditioning Using Busulfan Enables Bone Marrow Cell Accumulation in the Spinal Cord of a Mouse Model of Amyotrophic Lateral Sclerosis.</a> PLoS ONE. 8 (4), e60661 (2013).</li><li>Capotondo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+conditioning+is+instrumental+for+successful+microglia+reconstitution+following+hematopoietic+stem+cell+transplantation.">Brain conditioning is instrumental for successful microglia reconstitution following hematopoietic stem cell transplantation.</a> Proceedings of the National Academy of Sciences. 109 (37), 15018-15023 (2012).</li><li>Wilkinson, F. L., Sergijenko, A., Langford-Smith, K. J., Malinowska, M., Wynn, R. F., Bigger, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Busulfan+Conditioning+Enhances+Engraftment+of+Hematopoietic+Donor-derived+Cells+in+the+Brain+Compared+With+Irradiation.">Busulfan Conditioning Enhances Engraftment of Hematopoietic Donor-derived Cells in the Brain Compared With Irradiation.</a> Molecular Therapy. 21 (4), 868-876 (2013).</li><li>Lampron, A., Pimentel-Coelho, P. M., Rivest, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+bone+marrow-derived+cells+into+the+CNS+in+models+of+neurodegeneration:+Naturally+Occurring+Migration+of+BMDC+into+the+CNS.">Migration of bone marrow-derived cells into the CNS in models of neurodegeneration: Naturally Occurring Migration of BMDC into the CNS.</a> Journal of Comparative Neurology. , 3863-3876 (2013).</li><li>Kierdorf, K., Katzmarski, N., Haas, C. A., Prinz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+Marrow+Cell+Recruitment+to+the+Brain+in+the+Absence+of+Irradiation+or+Parabiosis+Bias.">Bone Marrow Cell Recruitment to the Brain in the Absence of Irradiation or Parabiosis Bias.</a> PLoS ONE. 8 (3), e58544 (2013).</li><li>Ajami, B., Bennett, J. L., Krieger, C., McNagny, K. M., Rossi, F. M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infiltrating+monocytes+trigger+EAE+progression,+but+do+not+contribute+to+the+resident+microglia+pool.">Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool.</a> Nature Neuroscience. 14 (9), 1142-1149 (2011).</li><li>Down, J. D., Tarbell, N. J., Thames, H. D., Mauch, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syngeneic+and+allogeneic+bone+marrow+engraftment+after+total+body+irradiation:+dependence+on+dose,+dose+rate,+and+fractionation.">Syngeneic and allogeneic bone marrow engraftment after total body irradiation: dependence on dose, dose rate, and fractionation.</a> Blood. 77 (3), 661-669 (1991).</li><li>Van Pel, M., van Breugel, D. W. J. G., Vos, W., Ploemacher, R. E., Boog, C. J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+myeloablative+regimen+with+clinical+potential:+I.+Treosulfan+conditioning+and+bone+marrow+transplantation+allow+induction+of+donor-specific+tolerance+for+skin+grafts+across+full+MHC+barriers.">Towards a myeloablative regimen with clinical potential: I. Treosulfan conditioning and bone marrow transplantation allow induction of donor-specific tolerance for skin grafts across full MHC barriers.</a> Bone Marrow Transplantation. 32 (1), 15-22 (2003).</li><li>Ajami, B., Bennett, J. L., Krieger, C., Tetzlaff, W., Rossi, F. M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+self-renewal+can+sustain+CNS+microglia+maintenance+and+function+throughout+adult+life.">Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.</a> Nature Neuroscience. 10 (12), 1538-1543 (2007).</li><li>Andersson, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioning+therapy+with+intravenous+busulfan+and+cyclophosphamide+(IV+BuCy2)+for+hematologic+malignancies+prior+to+allogeneic+stem+cell+transplantation:+a+phase+II+study.">Conditioning therapy with intravenous busulfan and cyclophosphamide (IV BuCy2) for hematologic malignancies prior to allogeneic stem cell transplantation: a phase II study.</a> Biology of Blood and Marrow Transplantation. 8 (3), 145-154 (2002).</li><li>Hassan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+and+plasma+concentrations+of+busulfan+during+high-dose+therapy.">Cerebrospinal fluid and plasma concentrations of busulfan during high-dose therapy.</a> Bone Marrow Transplantation. 4 (1), 113-114 (1989).</li><li>Hassan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+distribution+of+[11C]-busulfan+in+cynomolgus+monkey+and+in+the+brain+of+a+human+patient.">In vivo distribution of [11C]-busulfan in cynomolgus monkey and in the brain of a human patient.</a> Cancer Chemotherapy and Pharmacology. 30 (2), 81-85 (1992).</li><li>Jung, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Fractalkine+Receptor+CX3CR1+Function+by+Targeted+Deletion+and+Green+Fluorescent+Protein+Reporter+Gene+Insertion.">Analysis of Fractalkine Receptor CX3CR1 Function by Targeted Deletion and Green Fluorescent Protein Reporter Gene Insertion.</a> Molecular and Cellular Biology. 20 (11), 4106-4114 (2000).</li><li>Harrison, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competitive+repopulation:+a+new+assay+for+long-term+stem+cell+functional+capacity.">Competitive repopulation: a new assay for long-term stem cell functional capacity.</a> Blood. 55 (1), 77-81 (1980).</li><li>Evans, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+intravital+imaging+reveals+that+blood-derived+macrophages+but+not+resident+microglia+facilitate+secondary+axonal+dieback+in+traumatic+spinal+cord+injury.">High-resolution intravital imaging reveals that blood-derived macrophages but not resident microglia facilitate secondary axonal dieback in traumatic spinal cord injury.</a> Experimental Neurology. 254, 109-120 (2014).</li></ol>

The authors have nothing to disclose.

Condizionata busulfan consente la generazione di alto livello BM chimerismo in topi con cellule ematopoietiche che sono facilmente rilevabili. Questo condizionamento può essere eseguita senza la necessità di impianti di irradiazione. Inoltre, mielosoppressione con le dosi di busulfan descritto in questo protocollo è ben tollerato, riducendo al minimo gli effetti collaterali tossici causati da dosi mieloablativi di irradiazione, e quindi può essere una tecnica più adatta a generare BM chimerismo nei giovani, vecchi,...

Recently, there has been considerable interest in the roles of microglia and bone marrow (BM)-derived cells (BMDCs) in the central nervous system (CNS), both during disease as well as with normal aging. Microglia, the resident immune cells of the CNS, are now known to develop in the CNS following the entry of primitive myeloid progenitors during embryogenesis1. Microglia retain aspects of their myelomonocytic lineage well into adult life. While evidence suggests that the contribution of BMDCs to the microglial pool is minimal in healthy adult animals1, the role BMDCs play in the progression of various neurodegenerative diseases remains unclear. This uncertainty is compounded by the fact that it is difficult to distinguish BMDCs that accumulate within the CNS from endogenous resident microglia, as no universally accepted discriminating immunohistochemical marker has been identified. In order to monitor BMDCs in vivo, we employ a BM transplantation (BMT) protocol whereby endogenous BM cells of a recipient mouse are replaced with those of a donor mouse ubiquitously expressing green fluorescent protein (GFP) under control of the &#946;-actin promoter. This protocol permits the determination of both the localization and morphology of GFP+ BMDCs within the recipient mouse using immunohistochemistry, and facilitates further characterization of these cells using fluorescence-activated cell sorting (FACS) followed by subsequent biochemical assays. 
For a successful BMT, recipient BM cells need to be ablated (termed myeloablation) in order to generate niche space within the recipient BM to allow for donor cell engraftment. Often, myeloablation is achieved using total body gamma irradiation which induces double strand breaks in DNA that leads to cell death, particularly in actively dividing cells such as hematopoietic progenitor cells2,3. The irradiation protocols are done to induce sufficient BM cell death such that animal lethality results if the recipient animal does not achieve adequate donor cell engraftment from BMT (so called &#8216;lethal irradiation&#8217;). However, irradiation requires a specialized facility and equipment, along with veterinary and animal husbandry resources not available to researchers at all institutions. Furthermore, myeloablative irradiation can cause potentially lethal damage to other tissues, and due to immunosuppresion, irradiated animals are more susceptible to secondary infections3. As such, reduced intensity conditioning (so called &#8216;RIC&#8217; protocols) regimens have been developed that are intended to minimize potentially toxic side effects of the conditioning protocol in patients, particularly for use in at risk populations such as children and the elderly4. 
Some RIC protocols rely upon chemotherapeutics such as busulfan in order to condition the BM compartment. Busulfan is a bifunctional DNA alkylating agent often used clinically as an alternative to irradiation5,6. Notably, busulfan can be safely administered to mice by intraperitoneal (IP) injection and does not require the specialized facilities and equipment necessary to irradiate mice. Busulfan conditioning has been used extensively in our lab7, as well as in several other recent publications8&#8211;11. When doses of 60-100 mg/kg are employed, high degrees of stable chimerism (>80% GFP+ cells) can be established in the peripheral blood and BM7. Importantly, at these doses myeloablation is not complete and as such mice are able to survive without receiving support BM cells (K. Peake, J. Manning, C. Lewis, and C. Krieger, unpublished observations). Moreover, with myelosuppressive busulfan conditioning there is rapid reconstitution of peripheral blood myelomonocytic cells by donor cells. However, the replacement of peripheral blood lymphocytes by donor cells is slower, highlighting the lack of immunosuppression that occurs with 60-100 mg/kg doses of busulfan compared to total body irradiation7. 
We have successfully used busulfan-induced chimerism to investigate BMDCs in wild-type mice, as well as in mouse models of the neurodegenerative disorders amyotrophic lateral sclerosis (ALS)7 and Alzheimer disease. It has been observed that under certain conditions significant numbers of GFP+ BMDCs accumulate within the CNS7,12. Importantly, like myeloablative conditioning with irradiation, busulfan conditioning followed by BMT is sufficient to allow for GFP+ BMDC accumulation within the CNS in both wild-type mice, and mice with neurological disorders7&#8211;11. As a major obstacle in treating neurodegenerative disorders is the ability to get therapeutic molecules from the circulation into the CNS where they can exert beneficial effects, this raises the possibility that BMDCs could be engineered to express therapeutic molecules, and subsequently used as a vehicle to deliver therapeutics to the CNS of busulfan conditioned recipients, a mechanism we are actively investigating. Although we have found that myeloablative conditioning with irradiation leads to greater accumulation of GFP+ BMDCs in the CNS compared to busulfan conditioning7, irradiation may have considerable toxicity in patients with neurodegenerative disorders and in murine models of CNS disease.
While we, and many others, have used BMT models to study BMDCs in neurodegenerative disorders, the ability to largely replace the BM compartment of a recipient mouse with a distinctly identifiable population of BM cells is an invaluable tool for studying various aspects of the hematopoietic system. This includes a wide array of research topics such as hematopoietic lineage development, leukemia, organ transplantation, graft-versus-host disease, and immunobiology.

<table border="0" cellpadding="0" cellspacing="0" width="726">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:224px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Busulfex</td>
			<td style="width:224px;">Otsuka Canada Pharmaceutical Inc.</td>
			<td style="width:119px;">DIN 02240602</td>
			<td>Dilute busulfan to 3 mg/mL with sterile PBS just prior to use. Busulfan is cytotoxic. Handle and dispose according to the MSDS and institutional guidelines.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ACK Lysing Buffer</td>
			<td style="width:224px;">Life Technologies</td>
			<td style="width:119px;">A10492-01</td>
			<td>Other lysis buffer could be substituted but incubation times may need to be adjusted accordingly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:224px;">Life Technologies</td>
			<td>12483-020</td>
			<td></td>
		</tr>
	</tbody>
</table>

busulfan as a myelosuppressive agent for generating stable high-level bone marrow chimerism in mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism&nbsp;>80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

La somministrazione di 60-100 mg / kg di busulfan è generalmente ben tollerato dai topi. Tuttavia, gli animali in genere perdere peso durante la fase di condizionamento (~ 5-10%) e, quindi, la dieta può essere integrata con gli ossequi, come semi di girasole irradiati per incoraggiare mangiare. Livelli di chimerismo di GFP + cellule&gt; 80% nel sangue periferico e BM dovrebbero essere sempre raggiungibili con 60-100 mg / kg busulfan 7. Il nostro laboratorio ha utilizzato con successo questo protocollo per g...

Watch this Scientific Journal Video about Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice at JoVE.com

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

Busulfan come mielosoppressiva agente per la generazione stabile ad alto livello di midollo osseo chimerismo nei topi

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

busulfan-as-myelosuppressive-agent-for-generating-stable-high-level

Research

JoVE Journal

Medicine

13.1K Views.  Simon Fraser University. We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.