Efectos del enriquecimiento ambiental en el desarrollo del SNC

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Una aproximación a estrategias neuroprotectoras y neurorescatadoras

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Efectos del enriquecimiento ambiental en el desarrollo del SNC

  1. 1. Efectos del enriquecimiento ambiental en el desarrollo del SNC Una aproximación a estrategias neuroprotectoras y neurorescatadoras Laboratorio de Neurociencia Clínica y Experimental (LaNCE) Euskal Herriko Unibertsitatea http://www.ehu.es/ehusfera/lance viernes 12 de noviembre de 2010
  2. 2. Efectos del enriquecimiento ambiental en el desarrollo del SNC Una aproximación a estrategias neuroprotectoras y neurorescatadoras Laboratorio de Neurociencia Clínica y Experimental (LaNCE) Euskal Herriko Unibertsitatea http://www.ehu.es/ehusfera/lance Enrike G. Argandoña viernes 12 de noviembre de 2010
  3. 3. Desarrollo cortical Predeterminado genéticamente Mediado por experiencia viernes 12 de noviembre de 2010
  4. 4. Efectos del entorno en el desarrollo Lamarck, Haeckel, Darwin and the giraffe viernes 12 de noviembre de 2010
  5. 5. Cambios mediados por la experiencia Ramon y Cajal Sherrington viernes 12 de noviembre de 2010
  6. 6. Cambios mediados por la experiencia viernes 12 de noviembre de 2010
  7. 7. Cambios mediados por la experiencia APRENDIZAJE SIMPLE Aprendizaje simple Eric Kandel Aplysia californica artes 16 de junio de 2009 Aprendizaje simple Eric Kandel Aplysia californica artes 16 de junio de 2009 Eric Kandel Aplysia californica ueves 18 de junio de 2009 viernes 12 de noviembre de 2010
  8. 8. Cambios mediados por la experiencia APRENDIZAJE SIMPLE Aprendizaje simple Eric Kandel Aplysia californica artes 16 de junio de 2009 Aprendizaje simple Eric Kandel Aplysia californica artes 16 de junio de 2009 Eric Kandel Aplysia californica ueves 18 de junio de 2009 APRENDIZAJE SIMPLE Aprendizaje simple Eric Kandel Aplysia californica martes 16 de junio de 2009 Aprendizaje simple Eric Kandel Aplysia californica martes 16 de junio de 2009 Eric Kandel Aplysia californica jueves 18 de junio de 2009 viernes 12 de noviembre de 2010
  9. 9. Cambios mediados por la experiencia APRENDIZAJE SIMPLE Aprendizaje simple Eric Kandel Aplysia californica artes 16 de junio de 2009 Aprendizaje simple Eric Kandel Aplysia californica artes 16 de junio de 2009 Eric Kandel Aplysia californica ueves 18 de junio de 2009 APRENDIZAJE SIMPLE Aprendizaje simple Eric Kandel Aplysia californica martes 16 de junio de 2009 Aprendizaje simple Eric Kandel Aplysia californica martes 16 de junio de 2009 Eric Kandel Aplysia californica jueves 18 de junio de 2009 Eric Kandel viernes 12 de noviembre de 2010
  10. 10. Cambios mediados por la experiencia APRENDIZAJE SIMPLE Aprendizaje simple Eric Kandel Aplysia californica artes 16 de junio de 2009 Aprendizaje simple Eric Kandel Aplysia californica artes 16 de junio de 2009 Eric Kandel Aplysia californica ueves 18 de junio de 2009 APRENDIZAJE SIMPLE Aprendizaje simple Eric Kandel Aplysia californica martes 16 de junio de 2009 Aprendizaje simple Eric Kandel Aplysia californica martes 16 de junio de 2009 Eric Kandel Aplysia californica jueves 18 de junio de 2009 Eric Kandel Aplysia californica viernes 12 de noviembre de 2010
  11. 11. Plasticidad sináptica TEMA IV MECANISMOS SINÁPTICOS DE PLASTICIDAD Previo a la experiencia, se forman las vías mediante: Axones alcanzan la estación relay Axones alcanzan capa IV cortical Se forman conexiones aleatorias Posteriormente se produce el desarrollo influido por la experiencia Muerte celular programada (neurotrofinas) Cambios en capacidad sináptica martes 16 de junio de 2009 viernes 12 de noviembre de 2010
  12. 12. Plasticidad sinápticaTEMA IV MECANISMOS SINÁPTICOS DE PLASTICIDAD Cambios en capacidad sináptica Reducción 50% Reasignación sináptica Convergencia sináptica Competencia sináptica Influencias modulatorias tes 16 de junio de 2009 viernes 12 de noviembre de 2010
  13. 13. MECANISMOS SINÁPTICOS DE PLASTICIDAD Cambios en capacidad sináptica Reducción 50% Reasignación sináptica Convergencia sináptica Competencia sináptica Influencias modulatorias Locus ceruleus (NA) Nucleos basales (ACh) martes 16 de junio de 2009 viernes 12 de noviembre de 2010
  14. 14. TEMA IV MECANISMOS SINÁPTICOS DE PLASTICIDAD La plasticidad sináptica es un fenómeno excitatorio (Glutamato) NMDA (bloqueado por Mg+ + y ligado a canal Ca++) AMPA Na+ viernes 12 de noviembre de 2010
  15. 15. Cambios mediados por la experiencia Incremento sinapsis/neurona Incremento actividad neuronal Incremento demanda metabólica Modificaciones arquitectura vascular viernes 12 de noviembre de 2010
  16. 16. Age Experiencemediatedchanges 4. week 1., 2. and 3. weeks 4., 5. and 6. weeks 7. and 8. weeks Precritical period Critical period Postcritical period Periodo crítico viernes 12 de noviembre de 2010
  17. 17. PERIODO CRÍTICO 3ª - 5ª semanas Age Experiencemediatedchanges 4. week 1., 2. and 3. weeks 4., 5. and 6. weeks 7. and 8. weeks Precritical period Critical period Postcritical period Periodo crítico viernes 12 de noviembre de 2010
  18. 18. Periodo crítico viernes 12 de noviembre de 2010
  19. 19. Sistema visualEstudio efectos de la experiencia viernes 12 de noviembre de 2010
  20. 20. Estudio efectos de la experiencia Modificaciones sobre las condiciones standard * Enriquecimiento ambiental * Empobrecimiento ambiental viernes 12 de noviembre de 2010
  21. 21. Privación visual Métodos invasivos Inyección TTX Sutura párpados Enucleación mono o bilateral Extirpación retina viernes 12 de noviembre de 2010
  22. 22. Privación visual Métodos no invasivos Cría en oscuridad Implantación lentillas opacas viernes 12 de noviembre de 2010
  23. 23. Descenso densidad celular Retraso maduración Anulación cierre periodo crítico Privación visual viernes 12 de noviembre de 2010
  24. 24. Cortical parameters viernes 12 de noviembre de 2010
  25. 25. Cortical parameters viernes 12 de noviembre de 2010
  26. 26. Cortical parameters viernes 12 de noviembre de 2010
  27. 27. Vascular density viernes 12 de noviembre de 2010
  28. 28. Vascular density viernes 12 de noviembre de 2010
  29. 29. rain Research 732 (1996) 43-51 p=o , 04 p=o,l p=o,oo2 p=o,ol 0 dpn 7 dpn 14 dpn 21 dpn 60 dpn Fg. ; ’401 p=o,9 p=o,5 p=o,19 p=o,oo12 1204 T-r T p,m at birth to 500 pm at 7 dpn, or 70% with very high statistical significance. The second postnatal week, from 7 to 14 dpn, an increase of 587 pm, or 18’ZO,was registered, but the difference was not statistically significant. At the third week there was a slight decrease (3%), from 587 to 568 p,m with no significance. Cortical thickness at 60 dpn was similar to that of 14 dpn, increasing 3Y0from 21 dpn to reach 587 pm, but without statistical significance (see Tables 1 and 2). 3.1.2. Dark-reared rats This behavior was similar in dark-reared rats, but there was a quantitative difference at each age, with dark-reared rats scoring lower at all the considered ages, except at 14 dpn. From birth to 7 dpn there was a statistically significant 50% increase in cortical thickness in dark-reared rats, from 300 to 443 pm. From 7 to 14 dpn the increase was 56Y0, from 443 to 691 pm, and was highly significant. From 14 to 21 dpn there was a drop from 691 to 525 (25%), which was very significant. From 21 dpn to 60 dpn there was an increase of 3%, similar to the one in controls, which was not significant and reached 543 pm (see Tables 1 and 2). At 7 dpn cortical thickness was 12% higher in controls, at 14 dpn it was 1870higher in dark-reared rats, at 21 dpn it was 13Y0higher in controls and at 60 dpn it was 870 lower in dark-reared rats. The difference was statistically significant at all ages except at 14 dpn, being very high at 21 and 60 dpn (see Table 1 and Fig. 4). 3.2. Vascular densi~ 3.2.1. Controls The density of vessels increased massively up to 21 dpn in controls; at this age it reached the maximum level with a very weak increase from 21 to 60 dpn. Vascular density increased 12% at the first week, from 44 to 50 vessels per 40000 kmz. From 7 to 14 dpn there was almost a 50Y0increase, from 50 to 73 vessels. At 21 dpn vascular density increased 54%, from 73 to 112 vessels. At 60 dpn vascular density was similar to that at 0 dpn 7 dpn 14 dpn 21 dpn 60 dpn Fg. ; ’401 p=o,9 p=o,5 p=o,19 p=o,oo12 1204 T-r T 100 80 1 60 40 20 0 0 dpn 7 dpn 14 dpn 21 dpn 60 dpn ‘c ~ ; 30 z I TT p=o,13 P=0,16 p=o . oo l p=o , oo l Odpn 7 dpn 14 dpn 21 dpn 60 dpn ! Darkreared ! Controls Fig. 4. Comparison of average measurements between dark-reared and control groups at each of the ages considered. Horizontal axes show the age of the animals, Vertical axes show: CT, cortical thickness in pm; VD, number of intersections between vessels and the overlying grid per 40000 pmz of visual cortex. Nrad, number of vertically oriented intra- cortical vascular trunks per mm of cortex. to 14 dpn, an increase of 587 pm, or 18’ZO,was registered, but the difference was not statistically significant. At the third week there was a slight decrease (3%), from 587 to 568 p,m with no significance. Cortical thickness at 60 dpn was similar to that of 14 dpn, increasing 3Y0from 21 dpn to reach 587 pm, but without statistical significance (see Tables 1 and 2). 3.1.2. Dark-reared rats This behavior was similar in dark-reared rats, but there was a quantitative difference at each age, with dark-reared rats scoring lower at all the considered ages, except at 14 dpn. From birth to 7 dpn there was a statistically significant 50% increase in cortical thickness in dark-reared rats, from 300 to 443 pm. From 7 to 14 dpn the increase was 56Y0, from 443 to 691 pm, and was highly significant. From 14 to 21 dpn there was a drop from 691 to 525 (25%), which was very significant. From 21 dpn to 60 dpn there was an increase of 3%, similar to the one in controls, which was not significant and reached 543 pm (see Tables 1 and 2). At 7 dpn cortical thickness was 12% higher in controls, at 14 dpn it was 1870higher in dark-reared rats, at 21 dpn it was 13Y0higher in controls and at 60 dpn it was 870 lower in dark-reared rats. The difference was statistically significant at all ages except at 14 dpn, being very high at 21 and 60 dpn (see Table 1 and Fig. 4). 3.2. Vascular densi~ 3.2.1. Controls The density of vessels increased massively up to 21 dpn in controls; at this age it reached the maximum level with a very weak increase from 21 to 60 dpn. Vascular density increased 12% at the first week, from 0 dpn 7 dpn 14 dpn 21 dpn 60 dpn Fg. ; ’401 p=o,9 p=o,5 p=o,19 p=o,oo12 1204 T-r T 100 80 1 60 40 20 0 0 dpn 7 dpn 14 dpn 21 dpn 60 dpn ‘c ~ ; 30 z I TT p=o,13 P=0,16 p=o . oo l p=o , oo l Odpn 7 dpn 14 dpn 21 dpn 60 dpn ! Darkreared ! Controls Fig. 4. Comparison of average measurements between dark-reared and control groups at each of the ages considered. Horizontal axes show the BRAIN RESEARCH ELSEVIER Brain Research 732 (1996) 43-51 Research report Effects of dark-rearing on the vascularization of the developmental rat visual cortex E.G. Argandoiia a’*,J.V. Lafuente b aDepartment of Nursing I, School of Nursing, Euskal Herriko Unibertsitatea – University of the Basque Country, E-48940 Leioa, Spain b Department of Neuroscience, School of Medicine, Euskal Herriko Unibertsitatea – University of the Basque Country, E-48940 Leioa, Spain Accepted 16 April 1996 Abstract Cerebral vascular density corresponds to metabolic demand, which increases in highly active areas. External inputs play an important role in the modeling and development of the visual cortex. Experience-mediated development is very active during the first postnatal month, when accurate simultaneous blood supply is needed to satisfy increased demand. We studied the development of visuaf cortex vascularization in relation to experience, comparing rats raised in darkness with rats raised in standard conditions. The parameters measured were cortical thickness, vascular density and number of perpendicular vessels, constituting the first stage of cortical vascular development. Vessels were stained using butyryl cholinesterase histochemis~, which labels some neurons and microvascularization (vesselsfrom5 to 50 km). Animalsfromboth groupswere sampledat O,7, 14,21 and 60 days postnatal. Vascularization of the brain starts with vertically oriented intracortical vascular trunks whose density decreases notably after birth in rats reared in standard laboratory conditions. The most striking finding of our work is the significantly lower decrease in the number of these vessels in dark-reared rats. Our results also show that cortex thickness and vessel density are significantly lower in dark-reared rats. These results suggest that the absence of visual stimuli retards the maturation of the visuaf cortex including its vascular bed. Keywords: Striate cortex; Microvascularization; Development; Butyryl cholinesterase; Histochemistry 1. Introduction The density of the vascular network corresponds to the metabolic demand in different brain territories, with the demand increasing in areas with higher synaptic activity. Thus vascular density, and especially microvascular den- sity, becomes higher in these areas [6,7,23,34]. After birth, the neonate is exposed to external stimuli which modulate cortical development, inducing changes such as the increase of the dendritic tree, the rise of the ratio of synapses per neuron and the subsequent increase of the vascular network. All these changes involve the thickening of the cortex [14,34,35], Most of the cortical changes induced by experience occur in what is known as the critical period [16], which takes place around the 3rd postnatal week [1,24,30]. In this period, the higher metabolic demand due to neuronal plasticity mechanisms gives art extremely important role to adaptive vascular changes [34]. That is, the external environment induces vascular changes by an indirect mechanism: cortical devel- opment brought about by experience induces vascular plas- ticity to support the increased metabolism. Under standard rearing conditions blood vessels are essentially completely developed by the critical period [30]. Although this kind of change takes place all over the CNS, most studies of the effects of external inputs have been performed on the striate cortex. The visual system has a well-defined hierarchical organization which facili- tates the study of its structures through the interruption of pathways at different stages or the deprivation of inputs using either invasive techniques – tetrodotoxin injection Privación visual viernes 12 de noviembre de 2010
  30. 30. Privación visual .Brain Research 855 2000 137–142 www.elsevier.comrlocaterbres Research report Influence of visual experience deprivation on the postnatal development of the microvascular bed in layer IV of the rat visual cortex Enrike G. Argandona a,) , Jose V. Lafuente b ˜ a Department of Nursing I, School of Nursing, Euskal Herriko UnibertsitatearUniÕersity of the Basque Country, Leioa, E-48940, Spain b Department of Neuroscience, School of Medicine, Euskal Herriko UnibertsitatearUniÕersity of the Basque Country, Leioa, E-48940, Spain Accepted 16 November 1999 Abstract Cerebral vascular density is correlated with metabolic demands, which increase in highly active brain areas. External inputs are an essential requirement in the modeling of the visual cortex. Experience-mediated development is very active during the first postnatal month, when congruous blood supply is needed. We studied the development of visual cortex vascularization in relation to experience, comparing rats raised in darkness with rats reared in normal conditions. Vascular density, vascular area and their ratio vs. neuronal density were calculated. Conventionally stained semi-thin sections were used to measure the vascular area by computer assisted .morphometry. Animals from both groups were sampled at 14, 21, and 60 days postnatal dpn . We found a significantly lower density of vessels and neurons as well as a smaller vascular area in dark-reared adult rats while no differences were founded at the other ages. Our results also show no differences between the ratio of vesselsrneuron, and vascular arearneuron, between both groups. The absence of visual experience causes decrease of cortical activity which correlates with lower vessels density and vascular area, without their ratiorneuron being affected. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Dark-rearing; Microvascularization; Blood vessel; Striatal cortex; Computer assisted morphometry; Synaptic activity 1. Introduction There is a close relationship between metabolic activity w xand vascular network in cerebral cortex 3,9,24 . The metabolic demand, which increases in areas with higher synaptic activity is matched by the density of blood vessels w xin these areas 5,6,16,21 . As we have previously reported, development of vascularization parallels cortical develop- ment. Our results showed a decrease in vascular density and a delay in the maturation of the microvascular pattern Changes in vascular density are related to the different stages of development. Most of the changes induced by experience occur during a defined time-window of postna- tal life called critical period. In this period, there is reorga- nization of the cortex correlated with experience, when w xnon-functioning neurons disappear 12,25 . Studies of neu- ronal density in animals reared in darkness have found a relative increase due to a decrease in the neuropil; how- ever, putative changes in neuronal population have not w xbeen addressed 2,10,23 . Žstudied for both dark-reared and control groups percentage of .increaserdecrease and statistical significance, p value Ž . Ž . Ž .Age period dpn Darkness % p Controls % p Increase of Õascular density 14–21 51 -0.001 36.4 0.07 21–60 y21.1 -0.001 20 0.001 Increase of neuronal density 14–21 y9.9 0.02 14.3 0.29 21–60 y21.9 -0.001 y13 -0.001 Increase of Õascular area 14–21 y20.6 0.3 28.7 0.2 21–60 25.9 0.01 10.7 0.2 Increase of number of Õessels per neuron 14–21 66.7 -0.001 0 0.4 21–60 0 0.9 66.7 -0.001 Decrease of Õascular area per neuron 14–21 y27.8 0.08 37.7 0.04 21–60 4.3 0.6 y2.6 0.67 Decrease of aÕerage Õascular area 14–21 23.7 0.03 45.8 0.05 21–60 2.1 0.8 26.6 0.03 60 days, there was an increase of 20% which was not statistically significant. In dark-reared rats, the number of vessels increased by 51% between 14 and 21 dpn and decreased by 21% from the 21 to 60 dpn period. Both variations were significant. Comparing both groups, at 14 dpn, vascular density in dark-reared rats was 7% higher and at 21 dpn it was 18% higher. On the other hand, at 60 dpn, vascular density was 22% higher in controls. Differences were statistically sig- Ž .nificant at 21 and 60 dpn. Fig. 1 . 3.2. Neuronal density The number of neurons per 2500 mm2 was similar in all ages in normal rats, being maximal at 21 dpn. Between 14 and 21 dpn, neuronal density increased by 14% but decreased between 21 and 60 dpn by 13%. As it happened with the vascular density, the second increase from 21 to 60 dpn was significant but not the former from 14 to 21 dpn. The changes in this parameter throughout postnatal development in the dark-reared group were the opposite of controls as there was a progressive decrease from 14 to 60 dpn. From 14 to 21 dpn, this parameter decreased by 10%, from 16 to 15 neurons per 2500 mm2 . The decrease was Ž .more noticeable from 21 to 60 dpn 22% , diminishing from 15 to 11 neurons. Both differences were statistically Fig. 1. Comparison of average measurements between dark-reared and control groups at each of the ages considered. Horizontal axes show the ! ! ! viernes 12 de noviembre de 2010
  31. 31. Privación visual Developmental Brain Research 141 (2003) 63–69 www.elsevier.com/locate/devbrainres Research report Visual deprivation effects on the s100b positive astrocytic population in the developing rat visual cortex: a quantitative study a , b c *˜Enrike G. Argandona , Marco L. Rossi , Jose V. Lafuente a Department of Nursing I, School of Nursing, Euskal Herriko Unibertsitatea/University of the Basque Country, Leioako Campusa, Leioa E-48940, Spain b Department of Neuropathology, Walton Centre for Neurology and Neurosurgery, Liverpool L9 7LJ, UK c Department of Neuroscience, School of Medicine, Euskal Herriko Unibertsitatea/University of the Basque Country, Leioa, E-48940 Spain Accepted 11 December 2002 Abstract After birth, exposure to visual inputs modulates cortical development, inducing numerous changes of all components of the visual cortex. Most of the cortical changes thus induced occur during what is called the critical period. Astrocytes play an important role in the development, maintenance and plasticity of the cortex, as well as in the structure and function of the vascular network. Dark-reared Sprague–Dawley rats and age-matched controls sampled at 14, 21, 28, 35, 42, 49, 56 and 63 days postnatal (dpn) were studied in order to elucidate quantitative differences in the number of positive cells in the striate cortex. The astrocytic population was estimated by immunohistochemistry for S-100b protein. The same quantification was also performed in a nonsensory area, the retrosplenial granular cortex. S-100b positive cells had adult morphology in the visual cortex at 14 dpn and their numbers were not significantly different in light-exposed and nonexposed rats up to 35 dpn, and were even higher in dark-reared rats at 21 dpn. However, significant quantitative changes were recorded after the beginning of the critical period. The main finding of the present study was the significantly lower astroglial density estimated in the visual cortex of dark-reared rats over 35 dpn as well as the lack of difference at previous ages. Our results also showed that there were no differences when comparing the measurements from a nonsensory area between both groups. This led us to postulate that the astrocytic population in the visual cortex is downregulated by the lack of visual experience. ! 2002 Elsevier Science B.V. All rights reserved. Keywords: Dark-rearing; Immunohistochemistry; Astroglia; Retrosplenial cortex; RSG 1. Introduction vascular density as well as a delay in the development of the microvascular pattern [2,3]. Postnatal development of the visual cortex is modulated Astrocytes play an important role in the maintenance of by experience. Extrinsic cues act as epigenetic factors in the structure and function of the endothelium of the concert with intrinsic developmental programmes to shape cortex’s microvascular network, including the blood–brain functional and structural cortical architecture [29]. Ex- barrier [8,20]. Astrocytes are also involved in the develop- perience-mediated changes induce an increase in neuronal ment, plasticity and maintenance of the cerebral cortical activity, which leads to increased metabolic demands architecture [10,23,24,27]. Therefore, a crucial role in [6,40], involving the establishment of adaptive changes to coupling neuronal activity to energy metabolism has been accomplish new requirements such as changes of the proposed [13]. Inversely, both neuronal activity and vas- vascular network [5,7]. In previous works, we reported the culature influence glial development in a bidirectional development of vascularisation of the visual cortex in manner [1,18,22,41]. normal and dark-reared rats showing a decrease in the During development, astrocytes guide cortical organisa- tion by performing different functions, which are reflected in morphological, electrophysiological and antigenic dif- ferences [22]. The behaviour of the glia throughout the*Corresponding author. Tel.: 134-94-601-5595; fax: 134-94-464- postnatal development of the cerebral cortex can be studied9511. ˜E-mail address: nfpguare@lg.ehu.es (E.G. Argandona). using several antigenic markers. The most frequently used 0165-3806/02/$ – see front matter ! 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-3806(02)00643-0 morphology of astrocytes. and star shaped processes. S-100b positive cells were Measurements of each slice of the cortex were made in widely present on all cortical layers in opposition to GFAP, both hemispheres, for each of the ten slices taken per which was almost absent in middle areas while only some animal (i.e. 60 fields per animal) and the mean value per isolated astrocytes were present in lower and upper areas. animal was calculated. The average values per group (eight Outside the cortex, both GFAP and S-100b were present, animals) were compared at each age by statistical analysis especially in regions such as the hippocampus (Figs. 1 and (ANOVA) on STATVIEW IIீ Abacus Concepts. 2). All animal experiments were performed in accordance Comparing both experimental groups at all ages, no with the European Community Council Directive of 24 morphological differences were found. Thus, we per- November 1986 (86/609/EEC). formed a quantitative analysis in order to study the possible differences in the number of astrocytes per area. The results are shown in Table 1. Quantifying the number 3. Results of positive cells per cortical surface, the following results were obtained. We found a significantly lower number of cells per unit area in the visual cortex of dark-reared rats at 35 dpn, 3.1. Visual cortex whereas no differences were found between both groups in younger animals, with the exception of 21 dpn, when The density of S-100b positive cells suffered slight Fig. 1. (a) S100b positivity throughout the visual cortex at 5 weeks postnatal in dark-reared rats. (b) S100b positivity throughout the visual cortex at 5 weeks postnatal in control rats. (c) GFAP positivity throughout the visual cortex at 5 weeks postnatal in dark-reared rats. Positive cells appear mostly in lower and upper layers being almost absent in middle layers. Scale line is 150 mm. Fig.2.S-100bpositivecellsinlayerIVofdarkrearedvisualcortex.Scalelineis75mm. increasesanddecreasesbetweenalltheagesstudied,butdecreaseswerequantitativelylowerinresultstakenfrom whenwecomparedthelastofthestudiedages—63dpn—layerIV,showingahigherlevelofhomogeneity.Compar- whiledensityincontrolswassimilartodensityat14dpn,ingbothgroups,densitywasslightlyhigherindark-reared itwas25%lowerindark-rearedrats.Increasesandratsupto28dpn,andwassignificantonlyat21dpn.From Table1 Astroglialdensityatvariousagesofdevelopmentforbothdark-rearedandcontrolgroupsinthevisualandretrosplenialcortex(meannumberofS-100b 2 positivebodiesper250000or10000mm6standarddeviationandstatisticalsignificance,Pvalue) 22 AgeAstrocytesper250000mm(middlelayers)Astrocytesper10000mm(layerIV) (days) DarkrearingControlsDiff.(%)PDarkrearingControlsDiff.(%)P Visualcortex 1446.269.445.9681.90.6216.064.116.263.81.80.86 2158.269.952.5610.210.90.0122.066.117.463.726.40.0001 2847.168.543.268.190.0617.964.316.764.87.20.23 3536.068.35067.12280.000113.263.816.862.6221.40.0001 4231.866.839.967.4220.30.000111.863.315.364.8222.90.0001 4933.969.648.1613.4229.50.000112.263.916.965.4227.80.0001 5637.36942.169.7211.40.00913.96415.463.629.740.03 6334.467.845.367.72240.000112.663.316.964.9227.80.0001 Retrosplenialcortex 1441.8610.448.668.4140.0413.462.817.263.622.20.002 2160.969.248.8614.424.80.00521.56615.863.8360.0005 2844.6610.739.868.7120.1416.964.515.56490.14 3543.56745.668.224.60.3915.264.417.663.613.60.05 4237.167.338.465.323.40.5914.763.612.962.9140.11 4946.5612.351.4612.429.50.0916.365.818.26710.40.17 5640.467.743610.326.70.315.463.415.963.43.1%0.59 6345.567.549.969.928.80.116.064.316.163.620.10.93 viernes 12 de noviembre de 2010
  32. 32. 20 30 40 50 60 p14 p21 p28 p35 p42 p49 p56 p63 Oscuridad Control densidad astroglial astr./250000µm2 viernes 12 de noviembre de 2010
  33. 33. Empobrecimiento ambiental Corteza somatosensorial Corteza barrel ratas. Afeitado vibrisas produce alteraciones morfologicas y fisiologicas viernes 12 de noviembre de 2010
  34. 34. Empobrecimiento ambiental * Privación olfativa * Privación auditiva Similares efectos al resto de sentidos, pero de mayor intensidad Un elemento común es la plasticidad compensatoria en los sentidos no empobrecidos Neuron, Vol. 46, 103–116, April 7, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.neuron.2005.02.016 Activity-Dependent Adjustments of the Inhibitory Network in the Olfactory Bulb following Early Postnatal Deprivation Armen Saghatelyan,1 Pascal Roux,2 environmental conditions. The continuous postnatal sup- ply of newborn inhibitory interneurons to the main olfac-Michele Migliore,3,5 Christelle Rochefort,1,6 tory bulb (MOB) offers an ideal system to study neu-David Desmaisons,1 Pierre Charneau,4 ronal adjustment regulated by sensory experiences.Gordon M. Shepherd,3 and Pierre-Marie Lledo1, * Progenitor cells originating from the subventricular zone1 Laboratory of Perception and Memory (SVZ) of the lateral ventricle first migrate tangentially toPasteur Institute the MOB, by way of the rostral migratory stream (RMS),Centre National de la Recherche and then migrate radially within MOB before they dif-Scientifique (URA 2182) ferentiate into local interneurons (Luskin, 1993; Lois and75015 Paris Cedex Alvarez-Buylla, 1994). It has been hypothesized that post-France natal neurogenesis is controlled by levels of sensory2 Platform of Dynamic Imaging activity (Frazier-Cierpial and Brunjes, 1989; Corotto etPasteur Institute al., 1994; Kirschenbaum et al., 1999; Saghatelyan et al.,25 rue du Dr. Roux 2003; Lledo et al., 2004). Hence, although proliferation75015 Paris Cedexviernes 12 de noviembre de 2010
  35. 35. Enriquecimiento ambiental Donald Hebb (1949) Kresh, Bennett, Rosenzweig, Diamond (60s) Combinación de complejidad de objetos inanimados y estimulación social. viernes 12 de noviembre de 2010
  36. 36. Enriquecimiento ambiental viernes 12 de noviembre de 2010
  37. 37. Enriquecimiento ambiental * Necesidad de estandarizar * Super- enriquecimiento * Rol del ejercicio viernes 12 de noviembre de 2010
  38. 38. Enriquecimiento ambiental viernes 12 de noviembre de 2010
  39. 39. Enriquecimiento ambiental viernes 12 de noviembre de 2010
  40. 40. Enriquecimiento ambiental viernes 12 de noviembre de 2010
  41. 41. Enriquecimiento ambiental Cambios anatómicos Plasticidad neuronal Sinaptogénesis Morfología sináptica Neurogénesis Neurotrofinas (BDNF, NGF, NT-3,VEGF) Gliogénesis viernes 12 de noviembre de 2010
  42. 42. Enriquecimiento ambiental * Corteza auditiva (Greenough, 1973) * Corteza olfatoria (Roselli-Austin, 1990) * Corteza somatosensorial (Coq, 1998) * Hipocampo (Rampon, 2000) * Amigdala (Nikolaev. 2002) * Ganglios basales (Comery, 1996) * Cerebelo (Greenough, 1986) viernes 12 de noviembre de 2010
  43. 43. Enriquecimiento ambiental Mejora aprendizaje y memoria (Dash, 2009) Reduce deterioro cognitivo fisiologico (Segovia, 2009) Reduce ansiedad e incrementa actividad exploratoria (Benaroya, 204) Induce neurogenesis en hipocampo (Kempermann 1997) Reduce comportamientos adictivos a drogas (Solinas 2010) Madura y consolida la corteza visual en ratas privadas de luz (Bertoletti 2004) Acelera el desarrollo de la corteza visual (Cancedda 2004) viernes 12 de noviembre de 2010
  44. 44. viernes 12 de noviembre de 2010
  45. 45. Qualitativestudy LEA EBA GluT-1 HistochemistryImmunohistochemistry viernes 12 de noviembre de 2010
  46. 46. LEA EBA Qualitativestudy viernes 12 de noviembre de 2010
  47. 47. Qualitativestudy EBA GluT-1 EBA + GluT-1 viernes 12 de noviembre de 2010
  48. 48. Enriquecimiento ambiental Angiogénesis viernes 12 de noviembre de 2010
  49. 49. Quantitativestudy viernes 12 de noviembre de 2010
  50. 50. Quantitativestudy viernes 12 de noviembre de 2010
  51. 51. VEGFlevels WESTERN BLOT ELISA viernes 12 de noviembre de 2010
  52. 52. Western blot viernes 12 de noviembre de 2010
  53. 53. Western blot viernes 12 de noviembre de 2010
  54. 54. ELISA viernes 12 de noviembre de 2010
  55. 55. VEGF levels 0 1,5 3,0 4,5 6,0 14 dpn 21 dpn 28 dpn 35 dpn 42 dpn 49 dpn 56 dpn 63 dpn CE Control DR DR-CE viernes 12 de noviembre de 2010
  56. 56. !"#$%&'"!"#$$%& '()*!+$,++++-.,+/0%1234$,#$$%,$++$5,6 Blackwell Publishing Ltd !"#$%&'()*+*,&%$*)%$),*-.%,*/)01,)*23%,124*25'() *2,%&"4*25)51)100$*5)5"*)-.'25%5'5%3*)*00*&5$)10) /',67,*',%28)12)5"*)97:;;ββββ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`!)A!=(=BC!'B>NA:??!I(>!=9:!'B>N1>:B>)AO :6F:>)E:A=?`!BA'!'B>N1>:B>)AO!)A!D(A')=)(A?!(I!:A>)D9:'!:AY)>(AE:A=![)=9(<=!BA'![)=9!F9H?)DBC!:6:>D)?:,!89: B?=>(DH=)D!':A?)=H![B?!:?=)EB=:'!XH!)EE<A(9)?=(D9:E)?=>H!I(>!G1+$$β ! !F>(=:)A,!a<BA=)I)DB=)(A?![:>:!F:>I(>E:'!)A ! CBH:>!LV,!89:!?(EB=(?:A?(>)BC!D(>=:6!XB>>:C!I):C'![B?!BC?(!?=<'):'!B?!D(A=>(C,!89:!Y(C<E:!(I!CBH:>!LV![B?!?=:>:(C(O)DBCCH DBCD<CB=:'!I(>!:BD9!>:O)(AQ!BO:!BA'!:6F:>)E:A=BC!D(A')=)(A,!b>(E!=9:!X:O)AA)AO!(I!=9:!D>)=)DBC!F:>)('Q!B?=>(DH=: ':A?)=H![B?!9)O9:>!)A!D(A=>(C!>B=?!=9BA!)A!=9:!:A>)D9:'!:AY)>(AE:A=!O>(<F![)=9(<=!F9H?)DBC!:6:>D)?:Q![)=9!':A?)=):? (I!B?=>(DH=:?!B>(<A'!#$c!9)O9:>!B=!BCC!(I!=9:!')II:>:A=!BO:?,!LA!D(A=>B?=Q![9:A!=9:!BA)EBC?!9B'!BDD:??!=(!Y(C<A=B>H :6:>D)?:Q!':A?)=):?![:>:!?)OA)I)DBA=CH!9)O9:>!=9BA!:Y:A!=9:!D(A=>(C!>B=?,!<>!EB)A!>:?<C=!?9([?!=9B=!?=>B=:O):?!=( BFFCH!:AY)>(AE:A=BC!:A>)D9E:A=!?9(<C'!BC[BH?!D(A?)':>!=9:!)AD(>F(>B=)(A!(I!F9H?)DBC!:6:>D)?:Q!:Y:A!I(>!?:A?(>)BC B>:B?!?<D9!B?!=9:!Y)?<BC!B>:BQ![9:>:!D(EFC:6!:A>)D9:'!:6F:>):AD:!XH!)=?:CI!)?!A(=!:A(<O9!=(!D(EF:A?B=:!=9:!:II:D=? (I!Y)?<BC!':F>)YB=)(A, ) >*#)?1,/$ ! B?=>(OC)B`!'B>N1>:B>)AO`!:AY)>(AE:A=BC!:A>)D9E:A=`!G1+$$β ! `!Y)?<BC!D(>=:6`![9::C!><AA)AO, ) @25,1/.&5%12 ! 89:!F(?=AB=BC!':Y:C(FE:A=!(I!=9:!Y)?<BC!D(>=:6!)?!E('<1 CB=:'!XH!:6F:>):AD:Q![9)D9!?9BF:?!I<AD=)(ABC!BA'!D(>=)DBC B>D9)=:D=<>:,!M6F:>):AD:1E:')B=:'!D9BAO:?!B>:!B=!B!EB6)E<E '<>)AO!B!F>:':=:>E)A:'!=)E:![)A'([!DBCC:'!=9:!D>)=)DBC F:>)('!"K:>B>')!:=!BC,!#$$$`!S:A?D9Q!#$$3&Q![9)D9!)A!=9:!>B= Y)?<BC!D(>=:6!)?!X:=[::A!=9:!=9)>'!BA'!I)I=9!F(?=AB=BC![::N [)=9!B!F:BN!B=!=9:!I(<>=9![::N!"bBO)(C)A)!:=!BC,!+%%/&,!G(E: B<=9(>?! 9BY:! ?=<'):'! =9:! :II:D=?! (I! =9:! )AD>:B?:! BA'-(> ':F>)YB=)(A!(I!Y)?<BC!:6F:>):AD:!(A!=9:!A:<>(ABC!"K:AA:== :=!BC,!+%0/`!dBAD:''B!:=!BC,!#$$/&Q!O:A:=)D!"eBEF(A!:=!BC, #$$$&Q!YB?D<CB>!"KCBDN!:=!BC,!+%42`!G)>:YBBO!:=!BC,!+%44` ;>OBA'(PB!f!WBI<:A=:Q!+%%0Q!#$$$`!;>OBA'(PB!:=!BC,!#$$3` K:AO(:=6:B!:=!BC,!#$$4&!BA'!B?=>(OC)BC!"d(>Y:==)!:=!BC,!#$$5Q !#!# viernes 12 de noviembre de 2010
  57. 57. 10 12 14 16 18 20 22 24 P21 P28 P35 P42 P49 P56 P63 Primaryvisualcortex (S-100Bpositiveastrocytedensity) Postnatal Age C DR DR-EE DR-EE-Ex DR-Ex viernes 12 de noviembre de 2010
  58. 58. 9 12 15 18 21 24 P21 P28 P35 P42 P49 P56 P63 Primarysomatosensorycortex (S100-Bpositiveastrocytedensity) Postnatal Age C DR DR-EE DR-EE-Ex DR-Ex viernes 12 de noviembre de 2010
  59. 59. Patología SNC TCE Ictus Tumores Patologías neurodegenerativas viernes 12 de noviembre de 2010
  60. 60. Patología SNC TCE Ictus Tumores Patologías neurodegenerativas Vascularización viernes 12 de noviembre de 2010
  61. 61. Objetivos terapeúticos Neuroprotección/neurorescate Incremento vascularización viernes 12 de noviembre de 2010
  62. 62. TCE en Desarrollo Mayor capacidad de plasticidad Interferencia en los mecanismos fisiológicos Apoptosis Plasticidad sináptica (NMDA) viernes 12 de noviembre de 2010
  63. 63. Reserva Cerebral Cognitiva (Nithianantharajah, 2006) viernes 12 de noviembre de 2010
  64. 64. Reserva Cerebral Cognitiva (Nithianantharajah, 2006) viernes 12 de noviembre de 2010
  65. 65. Reserva Cerebral Cognitiva (Nithianantharajah, 2006) viernes 12 de noviembre de 2010
  66. 66. Enfermedades neurodegenerativas Alzheimer: reduce deposito ß amiloide (Cracchiolo, 2007), facilita su eliminación (herring, 2008), mejora deterioro cognitivo (Levi, 2003) Hungtington: disminuye deterioro cognitivo (Hannan, 2008) Parkinson: aumenta resistencia MPTP, (Thiriet, 2008); reduce deterioro estriado (Bezard, 2003) S. Rett y Down reduce sintomas motores y cognitivos. (Martinez-Cue, 2005); (Kondo 2008) viernes 12 de noviembre de 2010
  67. 67. Isquemia Disminuye secuelas (Saucier, 2010) Facilita migracion celulas SVZ (Hicks, 2007) Mejora recuperación funcional (Briones, 2009) Disminuye amiloidogenesis (Briones, 2009) viernes 12 de noviembre de 2010
  68. 68. TCE Promueve recuperacion funcion cognitiva (Hamm, 1996) Reduce daño BHE (Ortuzar, 2010) Disminuye muerte neuronal y mejora angiogenesis (Ortuzar, 2010) Recuperacion en rehabilitacion postraumática (Penn, 2009) viernes 12 de noviembre de 2010
  69. 69. Tumores Volume 142, Issue 1, 9 July 2010, Pages 52-64 Article Environmental and Genetic Activation of a Brain- Adipocyte BDNF/Leptin Axis Causes Cancer Remission and Inhibition viernes 12 de noviembre de 2010
  70. 70. e 3 '()(&*+&,+-."+/,&'#0'&)(1+"2*#+.&#3/0( Rearing schedule viernes 12 de noviembre de 2010
  71. 71. viernes 12 de noviembre de 2010
  72. 72. VEGF infusion 18 dpn Long Evans rats Alzet minipumps for 1 week at a 1 µl /h rate. VEGF. 25 ng/ml. anti-VEGF. 25 µg/ml. PBS. Untreated rats. viernes 12 de noviembre de 2010
  73. 73. BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - ORIGINAL ARTICLE Combination of intracortically administered VEGF and environmental enrichment enhances brain protection in developing rats Naiara Ortuzar • Enrike G. Argandon˜a • Harkaitz Bengoetxea • Jose´ V. Lafuente Received: 8 September 2010 / Accepted: 24 September 2010 Ó Springer-Verlag 2010 Abstract Postnatal development of the visual cortex is modulated by experience, especially during the critical period. In rats, a stable neuronal population is only acquired after this relatively prolonged period. Vascular endothelial growth factor (VEGF) is the most important angiogenic factor and also has strong neuroprotective, neurotrophic and neurogenic properties. Similar effects have been described for rearing in enriched environments. Our aim is to investigate the vascular and neuronal effects of combining VEGF infusion and environmental enrich- ment on the visual cortex during the initial days of the critical period. Results showed that a small percentage of Cleaved Caspase-3 positive cells colocalized with neuronal markers. The lesion produced by the cannula implantation resulted in decreased vascular, neuronal and Caspase-3 positive cell densities. Rearing under enriched environment was unable to reverse these effects in any group, whereas VEGF infusion alone partially corrected those effects. A higher effectiveness was reached by combining both the procedures, the most effective combination being when enriched-environment rearing was introduced only after minipump implantation. In addition to the angiogenic effect of VEGF, applied strategies also had synergic neu- roprotective effects, and the combination of the two strat- egies had more remarkable effects than those achieved by each strategy applied individually. Keywords Critical period Á Enriched environment Á Neuroprotection Á Neurovascular unit Á VEGF Á Visual system Introduction The development of the central nervous system (CNS), and more specifically of the sensory systems, is modulated by experience. This leads to an increase in metabolic demand (Black et al. 1990) that is satisfied by the adaptive remodelling of the vascular network (Argandon˜a and Lafuente 1996, 2000). Postnatal development of the visual cortex occurs in two stages. The first is genetically pre- determined and the second modulated by experience. Most of the cortical changes induced by experience occur during the critical period (Hensch 2005). This time window is specific for each sensory cortex and when experience- mediated reorganization finishes, sensory functions reach maturity (Bengoetxea et al. 2008). In rats, the critical period for the visual system is located between the third and the fifth postnatal weeks and the maximum peak of experience-induced changes occurs during the fourth and the fifth weeks (Fagiolini et al. 1994; Fagiolini and Hensch 2000). During development, more than half of the initially formed neurons die by programmed cell death (PCD), which is of fundamental importance for the correct devel- opment of the CNS (Finlay 1992). PCD is highly regulated during development and is maintained under strict control N. Ortuzar (&) Á E. G. Argandon˜a Á H. Bengoetxea Á J. V. Lafuente Department of Neuroscience, Laboratory of Clinical and Experimental Neuroscience (LaNCE), Faculty of Medicine and Odontology, University of the Basque Country UPV/EHU, Barrio Sarriena s/n, E48940 Leioa, Spain e-mail: naiara.ortuzar@ehu.es E. G. Argandon˜a Department of Nursing I, University of the Basque Country UPV/EHU, Barrio Sarriena, E48940 Leioa, Spain J Neural Transm DOI 10.1007/s00702-010-0496-2 imary visual cortex images for quantified vascular, neuronal pase-3 positive cell densities. Sections were stained by holinesterase histochemistry (a, b), NeuN (c, d) and Cleaved Caspase-3 (e, f) immunohistochemistry. Densities were esti the optical dissector method. Scale bar = 100 lm (a, c, e) a (b, d, f) d EE enhances brain protection in rats logical conditions (Nithianantharajah and Hannan 2009). EE has strong effects on the plasticity of neural con- nections, especially in the visual cortex, where it has been demonstrated that rearing from birth in an enriched pocket was opened in the back for the osmotic minipump placement (Mod. 1007 D, Alzet, Cupertino, CA, USA). The brain infusion kit (Mod. Alzet Brain Infusion Kit III, Alzet) was fixed to the skull with cyanoacrylate and the Fig. 1 Rearing conditions. a Standard condition and b enriched environment 123 Fig. 4 Primary visual cortex images for quantified vascular, neuronal and Caspase-3 positive cell densities. Sections were stained by butyryl cholinesterase histochemistry (a, b), NeuN (c, d) and Cleaved Caspase-3 (e, f) immunohistochemistry. Densities were estimated by the optical dissector method. Scale bar = 100 lm (a, c, e) and 20 lm (b, d, f) VEGF and EE enhances brain protection in rats viernes 12 de noviembre de 2010
  74. 74. Neuronal density viernes 12 de noviembre de 2010
  75. 75. NeuronalDensity (Opticaldissector) viernes 12 de noviembre de 2010
  76. 76. NeuronalDensity (Opticaldissector) viernes 12 de noviembre de 2010
  77. 77. Densidad vascular 0 5.500 11.000 16.500 22.000 SC EA Lesion Lesion EA Lesion SC-EA Lesion EA-SC 20061 1844918.344 16.935 21.694 18.149 viernes 12 de noviembre de 2010
  78. 78. Densidad neuronal 0 25.000 50.000 75.000 100.000 SC EA Lesion Lesion EA Lesion SC-EA Lesion EA-SC 626426110162.642 67.016 90.813 82.161 viernes 12 de noviembre de 2010
  79. 79. Densidad Caspasa3 0 5.500 11.000 16.500 22.000 SC EA Lesion Lesion EA Lesion SC-EA Lesion EA-SC 16738 18802 20.254 14.459 19.680 21.110 viernes 12 de noviembre de 2010
  80. 80. www.slideshare.net/nfpguare www.ehu.es/ehusfera/lance eg.argandona@ehu.es Contacto viernes 12 de noviembre de 2010

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