BACKGROUND
loss of retinal ganglion cells  occurs in many pathological situations, and glaucoma is one of the common diseases that lead to rgc loss.
a common feature of glaucoma is elevation of intraocular pressure  that causes progressive axonal degeneration and loss of rgcs.
as acute glaucoma is characterised by rapid increase in iop, acute iop elevation paradigm in rodents has often been used to study acute iop elevation-induced retinal ischemic/reperfusion injury and the possible mechanisms underlying acute glaucoma-associated rgc injuries  <cit> .
it is known that immune responses can influence neuronal survival after injury.
whereas ample evidence pointed to a damaging effect of inflammatory responses of macrophages and t-cells after cns injury and in autoimmune diseases such as multiple sclerosis and experimental autoimmune encephalomyelitis   <cit> , t-cell dependent rgc protection in response to iop elevation has also been reported in eae-resistant fischer  <dig>  but not eae-vulnerable lewis rats  <cit> .
in our earlier study, we found that macrophages, which are another major component of the autoimmune system, responded differently to iop elevation or optic nerve  injury between f <dig> and lewis rats, and such differences led to different extents of rgc survival after iop elevation or on injury  <cit> .
macrophage activation in the eye by intravitreal injection of zymosan, a yeast wall preparation, protected rgcs after on injury whereas the same activation aggravated rgc loss following acute iop elevation  <cit> .
in addition, under the same condition of either on injury or acute iop elevation the same macrophage activation resulted in different extents of rgc survival or loss between f <dig> and lewis rats  <cit> .
phosphatidylinositol 3-kinase /akt and janus kinase  signal pathways are well known to mediate neuronal survival  <cit> .
recently we showed that on injury or acute iop elevation activates both pathways in the ganglion cell layer   <cit> .
however, our recent work also points to paradoxic actions of these two pathways in rgc survival under different pathological conditions.
though both on injury and acute iop elevation activate pi3k/akt and jak/stat pathways in the gcl, including rgcs, inhibition of these signalling pathways activates macrophages in the eye and contributes to rgc survival after on injury in f <dig> rats  <cit> .
however, the same pathway inhibition leads to rgc loss following acute iop elevation in sprague dawley  rats  <cit> .
in addition, as mentioned above, we also showed that macrophage activation in the eye by intravitreal injection of zymosan played a protective role in rgcs after on injury  <cit> .
but the same macrophage activation resulted in aggravated rgc loss in an iop elevation model of the same f <dig> rat strain  <cit> .
macrophages thus appear to play an important role in the differences in rgc viability under the two pathological conditions.
f <dig> and lewis rats are inbred strains.
they differ in susceptibility to eae.
it is known that the hypothalamic-pituitary-adrenal  axis modulates autoimmune response and vulnerability to eae.
in contrast to f <dig> rats, lewis rats have abnormalities in hpa function.
variation in genotypes that are associated with different disease phenotypes between f <dig> and lewis rats have been reported  <cit> .
furthermore, the greater frequency of cd8+ regulatory t cells, which functionally inhibit myelin basic protein-reactive t-cells, in f <dig> than in lewis rats might contribute to the differing susceptibility to eae between them  <cit> .
it is currently unknown whether, after acute iop elevation, 1) inhibition of the pi3k/akt or jak/stat pathway aggravates rgc loss in f <dig> and lewis rats, 2) autoimmune background influences pi3k/akt and jak/stat signaling pathways in rgc survival, and 3) differential actions of macrophages occur in pi3k/akt and jak/stat pathway-dependent modulation of rgc survival.
pi3k/akt and jak/stat pathways play an important role in mediating rgc survival following acute iop elevation in spd rats  <cit> .
autoimmune background is also known to modulate neuronal viability  <cit> .
it is therefore important to clarify the issues as mentioned above.
this study, using f <dig> and lewis rats, was designed to accomplish these tasks.
RESULTS
effect of inhibition of pi3/akt and jak/stat pathways on rgc viability in f <dig> rats
representative photomicrographs showing the appearances of fg-labelled viable rgcs  and ed1+ macrophages  in normal f <dig> and lewis rats or  <dig> weeks after acute iop elevation plus various experimental interventions are shown in figure  <dig>  the appearances of fg-labelled viable rgcs  <dig> weeks after acute iop elevation alone in both f <dig> and lewis rats were previously shown  <cit> .
compared with normal intact f <dig>  and lewis  rats, significant loss of rgcs was seen  <dig> weeks after acute iop elevation and pathway inhibition of pi3k/akt in both strains .
clodronate liposomes significantly reduced the number of macrophages in the eye of both strains and, compared with the same strain of rats not receiving clodornate liposomes, significantly enhanced the number of surviving rgcs.
however, the extent of surviving rgcs after macrophage removal was still below the intact control, especially in lewis rats.
similar observations were seen after pi3k/akt pathway inhibition by ly <dig> or jak/stat pathway inhibition by ag <dig> and jak inhibitor i .
more surviving rgcs were seen in the central than the peripheral region.
no clear change in the retinal thickness was observed before and after the acute iop elevation  <cit> .
the average numbers ± sem of surviving rgcs and ed1+ macrophages were  <dig> ± 125/retina and  <dig> ± 2/retina, respectively, in intact f <dig> rats .
the average numbers  of surviving rgcs in the retinas of the iop elevation only group  and the inhibitor carrier dmso group  were not significantly different from each other .
intravitreal application of dmso marginally increased the number of macrophages  in the eye .
ly <dig>  the negative control of ly <dig> and which contains a single atom substitution in the morpholine ring compared to ly <dig> and does not inhibit pi3k even at high concentrations, did not affect rgc survival, but substantially increased the number of macrophages in the eye .
compared with the negative control ly <dig> and dmso groups, significant decreases in rgc survival were observed after intravitreal application of pi3k/akt pathway inhibitor ly <dig> or ky <dig> .
concomitant with the decrease in rgc survival was a significant increase in the number of macrophages in the retina .
significant decrease in the number of surviving rgcs was also seen after intravitreal application of jak/stat pathway inhibitor ag <dig> or jak inhibitor i .
accompanying the decrease in rgc survival was a significant increase in the number of macrophages in the retina .
these observations are thus similar to what was seen after inhibition of pi3k/akt and jak/stat pathways in spd rats  <cit> .
our earlier studies using western blotting showed that the pathway inhibitors used at these concentrations effectively, though not completely, blocked collective signal transduction of the pathways  <cit> .
effects of macrophage removal on rgc viability in f <dig> rats
we applied a macrophage remover, clodronate liposomes, intravitreally to deplete macrophages in the eye  <cit> .
as expected, clodronate liposomes applied alone or in combination with ky <dig>  significantly reduced the number of macrophages in the eye to a level slightly higher than that in dmso group .
concomitant with this reduction of macrophages in the eye was significant improvement but not complete recovery in rgc survival in ky12420-treated f <dig> rats .
similarly, an increased number of surviving rgcs was also seen after co-application of clodronate liposomes with jak/stat pathway inhibitor ag <dig> .
the improvement but not complete recovery of rgc survival after macrophage removal in pi3k/akt or jak/stat pathway-inhibited eyes suggested that macrophages and the pathways modulated, in opposite directions, rgc survival after acute iop elevation in f <dig> rats.
effect of inhibition of pi3k/akt and jak/stat pathways on rgc viability in lewis rats
the average numbers ± sem of surviving rgcs and ed1+ macrophages were  <dig> ± 82/retina and  <dig> ± 1/retina, respectively, in intact lewis rats .
the average numbers  of surviving rgcs and ed1+macrophages in the retinas of iop elevation only  and dmso  groups were not significantly different from each other in lewis rats .
however, the number of surviving rgcs  <dig> weeks after acute iop elevation was significantly  lower in lewis than f <dig> rats .
ly <dig> also did not affect rgc survival although it marginally increased the number of macrophages in lewis rats .
even though the levels of rgc survival were already low in the negative control and dmso groups , further decrease in rgc survival was still seen after intravitreal application of pi3k/akt pathway inhibitor ly <dig> or ky <dig> .
concomitant with the decrease in rgc survival was a significant increase in the number of macrophages in the retina .
similarly, intravitreal applications of jak/stat pathway inhibitor ag <dig> or jak inhibitor i  also resulted in a significant decrease in the number of surviving rgcs and a significant increase in the number of macrophages in the retina .
effects of macrophage removal on rgc viability in lewis rats
similar to what occurred in f <dig> rats, clodronate liposomes applied alone  or co-applied with ky <dig>  or ag <dig>  significantly reduced the number of macrophages in the retina to a level lower than that in the dmso group .
concomitant with the reductions of macrophages in the eye was significant improvement but not complete recovery in rgc survival in ky12420- and ag490-treated lewis rats .
these findings suggested that pi3k/akt and jak/stat pathways and macrophages are involved in rgc viability in similar fashion to that in f <dig> rats following acute iop elevation.
to clarify whether the observed reduction of rgc counts after inhibitor application resulted from interfered transport of fg, we carried out another experiment in which both fg and immunohistochemical approaches were used to label rgcs of the same retinas and the numbers of rgcs between the  <dig> approaches were compared.
compared with the number of tuj1-immunostained rgcs of the same retinas, there were an average of 15%  decrease and 12%  increase in the numbers of fg-labeled rgcs in ky <dig> and ag <dig> treatment groups, repsectively.
the small differences between the  <dig> labelling approaches are not statistically significant, indicating that the intravitreal application of the pathway inhibitors does not influence the efficacy of the fg transport.
note that fg was applied  <dig> hours whereas the data presented below were obtained from rats that received fg  <dig> days after the administration of the pathway inhibitors.
the further delayed application of fg rendered the inhibitors less likely to affect the retrograde transport of fg.
discussion
in this study we investigated the roles of pi3k/akt and jak/stat pathways and macrophages in rgc viability after acute iop elevation in f <dig> and lewis rats, which are known to have different autoimmune profiles.
we show that in both rat strains, inhibition of pi3k/akt or jak/stat pathway reduces rgc survival and activates macrophages in the eye.
in addition, both macrophage activation and pi3k/akt and jak/stat pathways mediate rgc viability, in opposite directions, after acute iop elevation.
pi3k/akt and jak/stat are known to be the major signalling executors for neuronal survival  <cit> .
whereas pi3k/akt is the common signal transduction pathway underlying neurotrophin-induced biological actions, jak/stat is well-documented to be responsible for cytokine-induced effects  <cit> .
previously we showed that the pathway inhibitors applied at dosages as in this study significantly inhibited signal transduction of the respective pathways  <cit> .
the clear differences in rgc survival or in macrophage recruitment between eyes treated with ly <dig> and with its negative control ly <dig> following iop elevation in both f <dig> and lewis rats are similar to what was seen in spd rats  <cit> .
these results confirmed that the actions of ly <dig> on rgc viability and macrophage recruitment are dependent on pi3k/akt pathway-inhibition.
the persistent loss of rgcs after pi3k/akt or jak/stat pathway inhibition in the absence of ocular macrophages  in both strains further verify that these signal transduction pathways mediate rgc survival following acute iop elevation independent of influence of autoimmune background.
previously t-cells were shown to play a part in differential protection of rgcs following episcleral and limbal vein cauterization-induced iop elevation in f <dig> and lewis rats  <cit> .
in our earlier study, we showed that macrophage reactions to acute iop elevation were also different in rats with different autoimmune backgrounds  <cit> .
in the present study we demonstrated that in contrast to the actions on macrophages, autoimmune background did not modulate signal transduction pathways of pi3k/akt and jak/stat in rgc survival.
CONCLUSIONS
pi3k/akt and jak/stat pathway mediate rgc survival after acute iop elevation and autoimmune background does not influence the functional roles of these pathways.
in addition, pi3k/akt and jak/stat pathway inhibition-induced macrophage activation in the eye is detrimental to rgcs following acute iop elevation.

 loss of retinal ganglion cells occurs in many pathological situations, and glaucoma is one of the common diseases that lead to rgc loss. a common feature of glaucoma is elevation of intraocular pressure that causes progressive axonal degeneration and loss of rgcs. as acute glaucoma is characterised by rapid increase in iop, acute iop elevation paradigm in rodents has often been used to study acute iop elevation-induced retinal ischemic/reperfusion injury and the possible mechanisms underlying acute glaucoma-associated rgc injuries. it is known that immune responses can influence neuronal survival after injury. whereas ample evidence pointed to a damaging effect of inflammatory responses of macrophages and t-cells after cns injury and in autoimmune diseases such as multiple sclerosis and experimental autoimmune encephalomyelitis, t-cell dependent rgc protection in response to iop elevation has also been reported in eae-resistant fischer but not eae-vulnerable lewis rats. in our earlier study, we found that macrophages, which are another major component of the autoimmune system, responded differently to iop elevation or optic nerve injury between f and lewis rats, and such differences led to different extents of rgc survival after iop elevation or on injury. macrophage activation in the eye by intravitreal injection of zymosan, a yeast wall preparation, protected rgcs after on injury whereas the same activation aggravated rgc loss following acute iop elevation. in addition, under the same condition of either on injury or acute iop elevation the same macrophage activation resulted in different extents of rgc survival or loss between f and lewis rats. phosphatidylinositol 3-kinase /akt and janus kinase signal pathways are well known to mediate neuronal survival. recently we showed that on injury or acute iop elevation activates both pathways in the ganglion cell layer. however, our recent work also points to paradoxic actions of these two pathways in rgc survival under different pathological conditions. though both on injury and acute iop elevation activate pi3k/akt and jak/stat pathways in the gcl, including rgcs, inhibition of these signalling pathways activates macrophages in the eye and contributes to rgc survival after on injury in f rats. however, the same pathway inhibition leads to rgc loss following acute iop elevation in sprague dawley rats. in addition, as mentioned above, we also showed that macrophage activation in the eye by intravitreal injection of zymosan played a protective role in rgcs after on injury. but the same macrophage activation resulted in aggravated rgc loss in an iop elevation model of the same f rat strain. macrophages thus appear to play an important role in the differences in rgc viability under the two pathological conditions. f and lewis rats are inbred strains. they differ in susceptibility to eae. it is known that the hypothalamic-pituitary-adrenal axis modulates autoimmune response and vulnerability to eae. in contrast to f rats, lewis rats have abnormalities in hpa function. variation in genotypes that are associated with different disease phenotypes between f and lewis rats have been reported. furthermore, the greater frequency of cd8+ regulatory t cells, which functionally inhibit myelin basic protein-reactive t-cells, in f than in lewis rats might contribute to the differing susceptibility to eae between them. it is currently unknown whether, after acute iop elevation, 1) inhibition of the pi3k/akt or jak/stat pathway aggravates rgc loss in f and lewis rats, 2) autoimmune background influences pi3k/akt and jak/stat signaling pathways in rgc survival, and 3) differential actions of macrophages occur in pi3k/akt and jak/stat pathway-dependent modulation of rgc survival. pi3k/akt and jak/stat pathways play an important role in mediating rgc survival following acute iop elevation in spd rats. autoimmune background is also known to modulate neuronal viability. it is therefore important to clarify the issues as mentioned above. this study, using f and lewis rats, was designed to accomplish these tasks.  effect of inhibition of pi3/akt and jak/stat pathways on rgc viability in f rats representative photomicrographs showing the appearances of fg-labelled viable rgcs and ed1+ macrophages in normal f and lewis rats or weeks after acute iop elevation plus various experimental interventions are shown in figure the appearances of fg-labelled viable rgcs weeks after acute iop elevation alone in both f and lewis rats were previously shown. compared with normal intact f and lewis rats, significant loss of rgcs was seen weeks after acute iop elevation and pathway inhibition of pi3k/akt in both strains. clodronate liposomes significantly reduced the number of macrophages in the eye of both strains and, compared with the same strain of rats not receiving clodornate liposomes, significantly enhanced the number of surviving rgcs. however, the extent of surviving rgcs after macrophage removal was still below the intact control, especially in lewis rats. similar observations were seen after pi3k/akt pathway inhibition by ly or jak/stat pathway inhibition by ag and jak inhibitor i. more surviving rgcs were seen in the central than the peripheral region. no clear change in the retinal thickness was observed before and after the acute iop elevation. the average numbers ± sem of surviving rgcs and ed1+ macrophages were ± 125/retina and ± 2/retina, respectively, in intact f rats. the average numbers of surviving rgcs in the retinas of the iop elevation only group and the inhibitor carrier dmso group were not significantly different from each other. intravitreal application of dmso marginally increased the number of macrophages in the eye. ly the negative control of ly and which contains a single atom substitution in the morpholine ring compared to ly and does not inhibit pi3k even at high concentrations, did not affect rgc survival, but substantially increased the number of macrophages in the eye. compared with the negative control ly and dmso groups, significant decreases in rgc survival were observed after intravitreal application of pi3k/akt pathway inhibitor ly or ky. concomitant with the decrease in rgc survival was a significant increase in the number of macrophages in the retina. significant decrease in the number of surviving rgcs was also seen after intravitreal application of jak/stat pathway inhibitor ag or jak inhibitor i. accompanying the decrease in rgc survival was a significant increase in the number of macrophages in the retina. these observations are thus similar to what was seen after inhibition of pi3k/akt and jak/stat pathways in spd rats. our earlier studies using western blotting showed that the pathway inhibitors used at these concentrations effectively, though not completely, blocked collective signal transduction of the pathways. effects of macrophage removal on rgc viability in f rats we applied a macrophage remover, clodronate liposomes, intravitreally to deplete macrophages in the eye. as expected, clodronate liposomes applied alone or in combination with ky significantly reduced the number of macrophages in the eye to a level slightly higher than that in dmso group. concomitant with this reduction of macrophages in the eye was significant improvement but not complete recovery in rgc survival in ky12420-treated f rats. similarly, an increased number of surviving rgcs was also seen after co-application of clodronate liposomes with jak/stat pathway inhibitor ag. the improvement but not complete recovery of rgc survival after macrophage removal in pi3k/akt or jak/stat pathway-inhibited eyes suggested that macrophages and the pathways modulated, in opposite directions, rgc survival after acute iop elevation in f rats. effect of inhibition of pi3k/akt and jak/stat pathways on rgc viability in lewis rats the average numbers ± sem of surviving rgcs and ed1+ macrophages were ± 82/retina and ± 1/retina, respectively, in intact lewis rats. the average numbers of surviving rgcs and ed1+macrophages in the retinas of iop elevation only and dmso groups were not significantly different from each other in lewis rats. however, the number of surviving rgcs weeks after acute iop elevation was significantly lower in lewis than f rats. ly also did not affect rgc survival although it marginally increased the number of macrophages in lewis rats. even though the levels of rgc survival were already low in the negative control and dmso groups, further decrease in rgc survival was still seen after intravitreal application of pi3k/akt pathway inhibitor ly or ky. concomitant with the decrease in rgc survival was a significant increase in the number of macrophages in the retina. similarly, intravitreal applications of jak/stat pathway inhibitor ag or jak inhibitor i also resulted in a significant decrease in the number of surviving rgcs and a significant increase in the number of macrophages in the retina. effects of macrophage removal on rgc viability in lewis rats similar to what occurred in f rats, clodronate liposomes applied alone or co-applied with ky or ag significantly reduced the number of macrophages in the retina to a level lower than that in the dmso group. concomitant with the reductions of macrophages in the eye was significant improvement but not complete recovery in rgc survival in ky12420- and ag490-treated lewis rats. these findings suggested that pi3k/akt and jak/stat pathways and macrophages are involved in rgc viability in similar fashion to that in f rats following acute iop elevation. to clarify whether the observed reduction of rgc counts after inhibitor application resulted from interfered transport of fg, we carried out another experiment in which both fg and immunohistochemical approaches were used to label rgcs of the same retinas and the numbers of rgcs between the approaches were compared. compared with the number of tuj1-immunostained rgcs of the same retinas, there were an average of 15% decrease and 12% increase in the numbers of fg-labeled rgcs in ky and ag treatment groups, repsectively. the small differences between the labelling approaches are not statistically significant, indicating that the intravitreal application of the pathway inhibitors does not influence the efficacy of the fg transport. note that fg was applied hours whereas the data presented below were obtained from rats that received fg days after the administration of the pathway inhibitors. the further delayed application of fg rendered the inhibitors less likely to affect the retrograde transport of fg. discussion in this study we investigated the roles of pi3k/akt and jak/stat pathways and macrophages in rgc viability after acute iop elevation in f and lewis rats, which are known to have different autoimmune profiles. we show that in both rat strains, inhibition of pi3k/akt or jak/stat pathway reduces rgc survival and activates macrophages in the eye. in addition, both macrophage activation and pi3k/akt and jak/stat pathways mediate rgc viability, in opposite directions, after acute iop elevation. pi3k/akt and jak/stat are known to be the major signalling executors for neuronal survival. whereas pi3k/akt is the common signal transduction pathway underlying neurotrophin-induced biological actions, jak/stat is well-documented to be responsible for cytokine-induced effects. previously we showed that the pathway inhibitors applied at dosages as in this study significantly inhibited signal transduction of the respective pathways. the clear differences in rgc survival or in macrophage recruitment between eyes treated with ly and with its negative control ly following iop elevation in both f and lewis rats are similar to what was seen in spd rats. these results confirmed that the actions of ly on rgc viability and macrophage recruitment are dependent on pi3k/akt pathway-inhibition. the persistent loss of rgcs after pi3k/akt or jak/stat pathway inhibition in the absence of ocular macrophages in both strains further verify that these signal transduction pathways mediate rgc survival following acute iop elevation independent of influence of autoimmune background. previously t-cells were shown to play a part in differential protection of rgcs following episcleral and limbal vein cauterization-induced iop elevation in f and lewis rats. in our earlier study, we showed that macrophage reactions to acute iop elevation were also different in rats with different autoimmune backgrounds. in the present study we demonstrated that in contrast to the actions on macrophages, autoimmune background did not modulate signal transduction pathways of pi3k/akt and jak/stat in rgc survival.  pi3k/akt and jak/stat pathway mediate rgc survival after acute iop elevation and autoimmune background does not influence the functional roles of these pathways. in addition, pi3k/akt and jak/stat pathway inhibition-induced macrophage activation in the eye is detrimental to rgcs following acute iop elevation. 
using an acute iop elevation paradigm in this study, we investigated 1) whether autoimmune background influences pi3k/akt and jak/stat functions by examining the effect of pi3k/akt and jak/stat pathway inhibition on rgc survival in f and lewis rats, and 2) whether differential actions of macrophages occur in pi3k/akt and jak/stat pathways-dependent modulation of rgc survival. in addition, the responses of macrophages to on injury and acute iop elevation were different between f and lewis rats, i.e., different autoimmune profiles. similar to what was previously observed, significantly more rgcs were lost in lewis than f rats weeks after acute iop elevation. this study demonstrates that following acute iop elevation 1) pi3k/akt and jak/stat pathways mediate rgc survival in both f and lewis rats, 2) autoimmune responses do not influence the functions of these two pathways, and 3) pi3k/akt and jak/stat pathway inhibition-dependent activation of macrophages is detrimental to rgcs. because macrophage invasion was seen in the eye after the pathway inhibition, to examine the role of these pathways independent of macrophages, macrophages in the retina were removed by intravitreal application of clodronate liposomes. removal of macrophages in the eye by clodronate liposomes reduced rgc loss due to pathway inhibition in both strains. viable rgcs were retrogradely labelled by fluorogold hours before animal sacrifice. as in spd rats, inhibition of the pi3k/akt or jak/stat pathway increased the loss of rgcs in both f and lewis rats. iop elevation was performed at mmhg for hours. we recently showed that whereas inhibition of pi3k/akt or jak/stat pathway promoted retinal ganglion cell survival after optic nerve injury in fischer rats, the same inhibition resulted in aggravated rgc loss after acute intraocular pressure elevation in sprague dawley rats. pi3k/akt and jak/stat pathway inhibitors were applied intravitreally to block their respective pathway signaling transduction.
 we recently showed that whereas inhibition of pi3k/akt or jak/stat pathway promoted retinal ganglion cell survival after optic nerve injury in fischer rats, the same inhibition resulted in aggravated rgc loss after acute intraocular pressure elevation in sprague dawley rats. in addition, the responses of macrophages to on injury and acute iop elevation were different between f and lewis rats, i.e., different autoimmune profiles. using an acute iop elevation paradigm in this study, we investigated 1) whether autoimmune background influences pi3k/akt and jak/stat functions by examining the effect of pi3k/akt and jak/stat pathway inhibition on rgc survival in f and lewis rats, and 2) whether differential actions of macrophages occur in pi3k/akt and jak/stat pathways-dependent modulation of rgc survival. iop elevation was performed at mmhg for hours. pi3k/akt and jak/stat pathway inhibitors were applied intravitreally to block their respective pathway signaling transduction. because macrophage invasion was seen in the eye after the pathway inhibition, to examine the role of these pathways independent of macrophages, macrophages in the retina were removed by intravitreal application of clodronate liposomes. viable rgcs were retrogradely labelled by fluorogold hours before animal sacrifice.  similar to what was previously observed, significantly more rgcs were lost in lewis than f rats weeks after acute iop elevation. as in spd rats, inhibition of the pi3k/akt or jak/stat pathway increased the loss of rgcs in both f and lewis rats. removal of macrophages in the eye by clodronate liposomes reduced rgc loss due to pathway inhibition in both strains.  this study demonstrates that following acute iop elevation 1) pi3k/akt and jak/stat pathways mediate rgc survival in both f and lewis rats, 2) autoimmune responses do not influence the functions of these two pathways, and 3) pi3k/akt and jak/stat pathway inhibition-dependent activation of macrophages is detrimental to rgcs. 