Document NGX5ZYvQJek7ZDor0QnREXm0y

Tot All Motors of tto Ls*d Bawlrocosotnl Bssltb Ccasltts* A*t Progress Rsport oo La-117 "Effect* of load oo Tlssus Culturs of tto Csatrsl fcrtcui Brstsa* -1 Attached for your lnforastlon ( * copy of tto report adteh ccwwrs tto work doos for us by Dr. Sobkovlct at Coluabls. I think you will flad :>5 tils quits interesting sad that It opees up teas lsportsot svmuss for futurs Investigation. Dr. Boblevies will prepare sa article for j*A>Uestlco In s nsdlcal Journal for sow futurs data. This srtlcls will, of courts, bs ssnt to us for review before It Is paMlstod. '3 Tsry truly yours. U/ DOfisw enclosure i Don 0. fowlsr Project Manager tSgS:;5> a^SfCKi  ' '! mcrasa REPORT Project |o; LH-117 Principal Inveetlgator j Advisor t - . Project Director: "Effect* of Lead oo Tissue Culture of the Central Bervou* Oystes" Dr. Margaret B. Murrey Professor of Anatomy Laboratory for 0*11 Physiology Columbia University College of Ihyalclana and Surgeon* Dr. Leonard J. Goldwater Profeaaor of Occupational Medicine School of Public Health and Administrative Medicine Columbia Uhlverelty Dr. Mann* M. Sofckowle* Assistant Profeaaor of Beurology Tissue Culture Laboratory of the Dept, of Beurology ~ University of Vlaconaln Medical School , An lnveatlgatlan of the direct action of lead ccopound* upon Serroua Tltauee in x-*j Culture m performed. The Method of tlaaue culture wee choeen for the study because of two neta advantage*: $ . 'I 1. The portion of the Dervou* eyetea under atudy can be expoeed directly to the experimental agent without the agent having been Metabolised by other organa. i 2. The blood-bra In-barrier vtth It* flltreticn function can be by-p*a*ed. Thu* through the Isolation of cultured tissue frta the whole organise, secondary effect* oo the nervous eyetea frc* poleonlrg of other organ* can be ellalnated. The lorg-tera culture* fro aeveral area* of tbe nervous eye tea cslnteln their cellular components with essentially noreal structure and function for conslderabl* periods (wets, Mentha). The culture* of central and peripheral nervajs system vere obtained fr an new born scus* and rat cerebellta and cerebral cortex, and frees tabrycnlc Mouse spinal cord and sensory gangll*. u n The study on the Influence of lead upon the nervous */*-* 1* concerned with three related but different preblea* considered eejeretely as Parts i I. II, and III, I, "Influence ft lead Acetate on the fve'.-Ear.t aad K 1 nt*rec-'_of hrvcai* Tissue To ixITVire'* (dl.H. Sot-kovictT fl.li. Mirray, T., jT^UuIivater, l.h. *Teteraon and KTnTlh rnaleln) for this atudy 417 culture* of newborn rat and Mouse ceretellum and spinal cord and eplnal ganglia ver* exposed to the heavy a*tel for perlM* of frrea 1 to 3 week*. The Influence of laad acetate In concentration* of frm 10*'M {0.J7) >/l) to 10'*M O'.w0 v/ml) of feeding solution was stalled for It* effect on Myelin Mlntenanct In nature culture* and on the pattern of development of yourg culture* ccnpared with those where no heavy seeial was present. jm*,. , eyjinpyjMptiaBipj N 477.01  ;h V# found no tvldane* of primary morphological change* In n*rv* cU bodlss, axon* or sheila *beath In culture* exposed to lend acetate In vitro, even in high concentration (37000 t/al of nutrient nedlu* up to one week of expoeure). There vu however a constant end marked degenerative reaction of macrophage* followed by hypertrophy of glial cell*. Stellar rerult* were obtained during preliminary exposure* of culture* to trlethyl tin sulfate and gold chloride. Culture* exposed to mercuric chloride (and thallium chloride and acetate - K.R. Pete non, M.R. Murray 1965) showed evidence of direct toxic action by these Betel* oo the nerve cell bodies or their axon*, with secondary change* in the Myelin sheath. The above reaction* of cultured nervous tissue see** to divide these Metal* Into two group*: l.- Thoee which cause a direct morphological injury to the nerve cell bodies or axons (aercurlc chloride, thallium chloride and acetate) and, 2.- Thoee which provoke morphological changes primarily In cell* of Mesodermal origin and only secondarily in neuroglia (lead acetate, trlethyl tin sulfate and gold chloride). Of the ertal* which were directly toxic to the nerve cell, the degree of toxicity was dependent oo the concentration. The action of metals which did not produce direct morphological response in the nerve cell wm* dependent mostly on the duration of exposure. Also the degeneration of ecto*and mesodermal supporting tissue was ccoaon to this group of metal*, lead, which was teited moct thoroughly, svto facilitated the development and differentiation of nervw* tlatue in young culture*, although the mechanics and meaning of thl* observation 1* not clear to us. Il.*Aborptlon of Lead Acetate by Hervou* Tissue in Culture" (H.H. Sobkovict, E.R. Petersen, M.ft Murray, L.J. CoIdwater) The lack of primary morphological change* in nerve cell*, their axons and myelin *hc*th* in culture* expose! to leal acetate, a* well a* the normal pattern cf the development and eye11nation of exposed young culture* (** - 6 day* In Vitro) va* rather intriguing to u*. Using the hlstochaslcal staining technique or rhcdltenlc acid for the pretence of lead we t tarted a systematic study of the up* take of lead acetate from the feeding solution by nervous tissues In Vitro. The mature myelinated cultures of new bora rat and mouse cerebellum and embryo mctite spinal cord and spinal ganglia were exposed to */0 *v/l of lead acetate in the feeding solution. As the results of cnr study ve showed that there is no evidence of the absorption of lead by the nerve cell In Vitro from le1-conl*l.V.ng medium. A heavy uptake of lead Is however observed in macrophages, aentgeal cells and to a lsss extent by the glial cells. Therefore it It slrorgly s-argested that the toxicological responses of nervous tissue culture* exposed-.to dead e.-elai* correspcoi to the hl*toch*lcal pattern of absorption of lead by the*#{Cultures, as we observe it. The lack of evidence of lead uptake frrm the nutrleM*'medium by tea ra-irjse eculi p'.*ln In fact th# rather surprising phenrmwncn of the Jack of toxicity of thl* metal to the nerve cell and fibre and the myelin sheath. Cm the other hand the cells which were Involved In the protest of leal absorption from live nutrient Belli* lhowe.1 buth toxic reaction *r.l sw.rphniglcel das*g# during the llae nf espoeure. Hi# appearor-'e of hyperlrophlei glial cell* a* - observed during repeated experiments alght be explained a* a secondary effect resulting frtss tie degenarait'vi of cacrop.hey.es and their possible llberattm of sure c-eplex, organic coejcajvl*. The rather Insignificant degree of lead absorption by the gllel cells, a* shown hlstochemlcaliy, doe* not seem t > Justify their susrked Involvement in the pathological proc*#* a* It 1* observed u> vitro. - /''',U'v ;'iulvrti _ / : . . / t *j ^u *m I ' * ve.Vx^/--W * tsw. Oi III. -3- "Influenc* of gerua frosi Laboratory Ant--la and Patient* with Lead PoUonlrst on Kervou* T1iu In Culture(H.M.&obkevict, k.R. Harray, L.J. Goldvaler t.ft. Poterton and K.fe. Beroe i* In). . LocVlrxj for * different approach to the problea of lead poisoning In vitro ' ' tested sera obtained fraa laboratory anlaals and patient* with lead poisoning, against nature nyallnated culture* of the nervous *y*tea. The exposure of the culture* to the** era reiulted In partial or cceplete degeneration and breakdown of the nye 1 In *heath, which In no*t lnatancea wma acccsspanied by eccplete preiervatlon of the nerve cell bodiea and axona, but not. of the glial cell* (which are equally Involved In lyelin oalntensr.es). Ve al*o demonstrated that the lyila and degeneration of central and peripheral ryelln In reapooaa to the preienca of the above aera la not correlated to the actual concentration of lead In the*# aera. Therefore It la poeatble that the oteerved Ryelln disturbance wa* not due to the actual pretence of lead but rather to other tubttancea aecondarlly present In theft acre. Our conclusions therefore are usnarlted aa follow* t ir -,=V. 1. There 1* evidence In vitro of central and peripheral aye1In degeneration and lyt* In re*ponte to the pretence of *erua frcn lead poisoned patient*, and laboratory anlaale. - ?. There 1* evidence that the degeneration of the Ryelln heath observed during exposure to lead poisoned aera l_n vitro wi not related quantitatively to the actual concentrations of lead In these sera. Thu* It It possible that the observed Ryelln disturbance* were not due to the pretence of the lead but rather to other diaturlar.ee* tecoodarlly operatic, (cf. Tart I). C! ' 3. Lead lteelf haa no direct toxic effect on the nerve ceil and the Ryelln sheath In vitro. This aay be due to the detacna(rated lack of uptake of the i Mill by the nerve cell fro* Ita envlronaent. (cf. Tart II) l. It la alto suggested that the excessive absorption of lead by Rseenchyaal tissue result* In the degeneration of thl* tta*u*. (cf. fart II) ' 3. A secondary response of the glia due to the degeneration of aetenchymal tissue, aa well aa It* noderate ability to ataorb leal Ui vitro suggests the Involveaent of IM* tissue In the patholcgy of lead poisoning In vitro. (cf. fart* 1 and II) 6. The hypothesis 1* proposed that the direct toxicity of leal toj^he nervous *yste Ui vitro 1* corrtUble with aaslallatlon of the aetal by It* varies** ccaponent tissue*. I j