Chem. Senses 26: 971-981,
2001
© Oxford University Press 2001
Volumetric and Horseradish Peroxidase Tracing Analysis of Rat Olfactory Bulb Following Reversible Olfactory Nerve Lesions
Center for Alzheimer Disease and Related Disorders, PO Box 19682, Southern Illinois University School of Medicine, Springfield, IL 62794, USA 1 Department of Biological Sciences, Eastern Illinois University, Charleston, IL 61920, USA
Correspondence to be sent to: Robert G. Struble, Center for Alzheimer Disease and Related Disorders. PO Box 19628, Southern Illinois University School of Medicine, Springfield, IL 62794-9628, USA. e-mail: rstruble{at}siumed.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Olfactory receptor neurons can regenerate from basal stem cells. Receptor neuron lesion causes degenerative changes in the olfactory bulb followed by regeneration as new olfactory receptor axons innervate the olfactory bulb. To our knowledge, parametric analyses of morphometric changes in the olfactory bulb during degeneration and regeneration do not exist except in abstract form. To better characterize olfactory bulb response, we performed morphometric analysis in rats following reversible olfactory nerve lesion with diethyldithiocarbamate. We also performed anterograde tracing of the olfactory nerve with wheatgerm agglutinin linked to horseradish peroxidase. Results of morphometry and tracing were complementary. The glomerular layer and external plexiform layer showed shrinkage of 45 and 26%, respectively, at 9 days. No significant shrinkage occurred in any other layer. Individual glomeruli shrank by 40-50% at 3 and 9 days following lesion. These data show that degenerative changes occur both in the glomeruli and transneuronally in the external plexiform layer. Olfactory nerve regeneration (identified by WGA-HRP transport) paralleled volumetric recovery. Recovery occurred first in ventral and lateral glomeruli between 9 and 16 days followed by recovery in medial and dorsal glomeruli. These data indicate substantial transynaptic degeneration in the olfactory bulb and a heretofore unrecognized gradient in olfactory nerve regeneration that can be used to systematically study recovery of a cortical structure.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The olfactory bulb (OB) presents a unique system for studying neuronal plasticity processes in the central nervous system. Numerous procedures have been described to lesion olfactory receptor neurons (ORN) which project to the glomerular layer of the olfactory bulb. Many of these procedures spare the basal stem cells that replace the injured ORN and reinnervate the OB (Margolis et al., 1974
; Harding et al.,
1977; Nadi et al.,
1981; Schwob et al.,
1995; Ravi et al.,
1997; Setzer and Slotnick,
1998). Changes occurring in the OB represent both responses to
degeneration and regeneration of the olfactory nerve terminals. Of major
significance is that degeneration and regeneration occur both in the terminal
fields of the olfactory nerves and transneuronally (see below).
Transneuronal processes in the OB are reasonably well known
(Pinching and Powell, 1972;
Margolis et al.,
1974; Nadi et al.,
1981; Baker et al.,
1983; Stone et al.,
1991; Sashihara et
al., 1997; Schwob et
al., 1999; Weruaga et
al., 2000). Electron microscopic evidence from rats showed
relatively minor changes (Pinching and
Powell, 1972). However, strikingly decreased OB expression of
tyrosine hydroxylase follows ORN lesion
(Nadi et al., 1981;
Baker et al., 1983;
Stone et al., 1991).
Several studies report elevation of glial markers not only around the
glomeruli, but in laminae that do not receive a direct input from the
olfactory nerve (Barber and Dahl,
1987; Anders and Johnson,
1990; Ravi et al.,
1997; Schwob et al.,
1999). Glial activation suggests that some neuropil reorganization
is occurring in these laminae, although the exact nature is cryptic. Growth
cone-associated protein in the olfactory bulb declines after lesion but is
elevated roughly 2-fold during regeneration
(Verhaagen et al.,
1990; Struble et al.,
1998; Schwob et al.,
1999). Amyloid precursor-like protein immunoreactivity initially
appears in degenerating ORN axons at 3 days after lesion, is then expressed by
glia at several days post-lesion and subsequently is expressed in dendrites of
neurons several weeks following the lesion
(Struble et al.,
1998). Similar processes probably occur in other parts of the CNS,
but are more difficult to visualize
(Cotman et al.,
1998). These processes are obvious in the OB because: (i) ORN
lesion removes a substantial part of the input to a single layer and (ii)
recovery after lesion extends for weeks because receptors must regenerate and
extend axons to the OB.
Distinct laminae with well-described cell types and connectivity further
facilitate interpretation of ORN lesions
(Greer, 1991). The olfactory
nerve layer (ONL) contains the axons of the ORN which terminate in the
glomerular layer (GL), contacting dendrites of mitral and tufted neurons
within the glomerulus. The external plexiform layer (EPL) does not receive ORN
axons, but is composed of processes of neurons post-synaptic to the ORN and
axons extrinsic to the OB. The mitral cell layer, which forms the inner limit
of the EPL, surrounds the internal plexiform layer (IPL) and the granule cell
layer (GCL). Granule cells have dendrites which extend to the EPL and engage
in reciprocal synapses with dendrites of mitral cells
(Rall et al., 1966).
Remnants of the ventricular layer and subependymal zone persist at the core of
the bulb. In juvenile animals this layer contains numerous precursor cells
that can differentiate into either glia or interneurons (see
Peretto et al.,
1999). This ability may persist into adulthood.
We were unable to find published volumetric analyses of the adult OB
following ORN lesion and during subsequent regeneration. These studies are
necessary for reasonable interpretation of the OB as a model for plasticity.
Therefore, in this study we have systematically evaluated the OB following a
reversible lesion of ORN. We combined volumetric analyses of the OB with
anterograde tracing studies of the olfactory nerve with wheatgerm
agglutinin-horseradish peroxidase (WGA-HRP). Relatively simple methods for
anterograde tracing of the olfactory nerve with WGA-HRP exist
(Shipley, 1985;
Stewart, 1985;
Baker and Spencer, 1986;
Itaya et al., 1987;
Yee and Costanzo, 1995;
Moon and Baker, 1998;
Setzer and Slotnick, 1998;
Schwob et al., 1999).
Hence, properly performed tracing studies can visualize the distribution of
regenerated ORN axons. We found transient shrinkage of both the GL and EPL
following olfactory nerve lesion, supporting transneuronal changes in the OB.
We also found that ventral and lateral glomeruli in the middle portions of the
OB recovered both innervation and size more rapidly than did medial or dorsal
glomeruli. These studies present important observations for utilization of the
OB as a model to study degeneration and regeneration in the central nervous
system.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
All procedures in these studies received prior approval by the Southern Illinois University School of Medicine Laboratory Animal Care and Use Committee. Rats were housed in the animal care facility, which is accredited by the AAALAC. They were maintained on a 12:12 light:dark cycle and food and water were available ad libitum.
ORN lesions
We performed reversible lesions of olfactory epithelium with
diethyldithiocarbamate (DDTC) as previously described
(Ravi et al., 1997;
Struble et al.,
1998). Wistar male rats (Harlan), 300-350 g body wt (60+ days of
age), were sedated with 7.6 ml/kg 5% chloral hydrate in distilled water. When
sedation was obtained, a 2.5 inch 20 gauge needle was inserted s.c. at the
intrascapular level and the rats were injected with 600 mg/kg DDTC dissolved
in normal saline (180 mg/ml). The skin surrounding the needle was pinched
closed and the needle withdrawn. Rats were killed at 3, 9, 16, 28 and 42 days
following DDTC injection.
WGA-HRP treatment
WGA-HRP (Sigma) solution was infused intranasally 48 h prior to death. Rats
were anaesthetized with 40 mg/kg pentobarbital and placed on their back.
Several differing concentrations of WGA-HRP and methods of applying WGA-HRP
via the external nares were tested. Our final technique used 50 µl of 4.0%
WGA-HRP (Sigma), diluted in 0.9% saline, slowly dripped into (1-2 mm) one
external naris with a pipette. The rats were allowed to inspire each drop
normally. During infusion (3-5 min) the rat was gently rolled from side to
side while still on its back. A 3.5 mm3 piece of gel foam (
1.5
mm/side), soaked in 4.0% WGA-HRP, was inserted 4-5 mm into the external
opening of the naris using an 18 gauge Abbocath (Abbott Laboratories)
catheter.
Tissue preparation
Forty-eight hours later the rats were deeply anesthetized with pentobarbital (80 mg/kg). When a pinch-withdrawal response was absent they were perfused with 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brain was removed and placed in fresh fixative in phosphate buffer for a further 2 h, cryoprotected overnight in 30% sucrose in phosphate buffer and frozen with dry ice. The turbinates were freed from the surrounding bone and post-fixed for 2 h, placed in Cal-Ex (Fisher Scientific) for 3 h and rinsed in phosphate buffer twice. They were then infiltrated with 3% warm gelatin, placed in 30% sucrose in phosphate buffer overnight and frozen with dry ice.
For quantitative analysis of laminar volume and glomerular area, a total of 21 DDTC-treated rats were used: four non-lesioned rats and survival periods of 3 (n = 3), 9 (n = 3), 16 (n = 4), 28 (n = 3) and 42 (n = 4) days. The OB was serially cut with a freezing microtome at 30 µm collected in phosphate buffer and every sixth section was directly mounted and dried on chrome alum subbed slides, defatted and stained with cresyl violet. For volumetric analysis the area of each lamina was outlined using Neurolucida (Colchester, VT), which combines an operator-generated overlay image on the microscopic image. Figure 1A shows a Nissl stained section with the laminae outlined. The area of the outlined area was automatically calculated. Volume was estimated by summing the area from two adjacent sections, dividing by two and multiplying by the interval (180 µm) between each section. To estimate area of glomeruli we measured the area of every glomerulus in one section roughly midway through the rostral-caudal axis of the OB (Figure 1A). This section was adjacent to the section used for quantification in tracing studies (see below). Glomeruli were operationally divided into four groups (dorsal, ventral, lateral and medial) and the area traced with Neurolucida. The operational definition of ventral (or dorsal) was made with a line perpendicular to the bottom (or top) of the ventricular layer.
|
WGA-HRP development
One in six OB sections was saved in phosphate buffer for WGA-HRP
development. Product was developed using a nickel intensification technique of
the HRP-diaminobenzidine (DAB) product
(Benzing and Mufson, 1995).
Briefly, sections were rinsed three times for 10 min each in 0.01 M imidazole
in 0.05 M acetate buffer (I/A buffer), adjusted to pH 7.4 with glacial acetic
acid. A 0.05% DAB solution was made in I/A buffer that had not been pH
adjusted. After sonication, nickel ammonium sulfate was added to make a 0.06 M
solution, then 1% H2O2 was added to make a 0.0015%
solution. Sections were reacted for 7 min then rinsed with buffered I/A,
mounted, dehydrated and cover-slipped.
Semi-quantification of glomerular labeling by WGA-HRP was performed on sections adjacent to those used for glomerular area measurements. For this analysis we used a total of 21 rats (five controls and three at 3, three at 9, four at 16, two at 28 and four at 42 days). In most cases these were the same rats as used in the volumetric studies. However, in several cases the quality of the WGA-HRP staining in rats used for morphometry was inadequate for analysis. Sections from two control rats were selected from a comparable rostro-caudal region to replace two controls of the morphometric analysis showing no WGA-HRP reactivity and one rat at 28 days showed no WGA-HRP reactivity. Glomerular staining with WGA-HRP was categorized in three groups. (i) Unstained glomeruli could be easily identified (when the substage condenser was stopped down) as having a clear core surrounded by somata. (ii) Partially stained glomeruli were defined as either lightly stained throughout the extent of the glomerulus or having patchy depositions of reaction product in the glomerulus. (iii) Fully stained glomeruli were defined as containing reaction product throughout the extent of the glomerulus. Figure 1B shows a WGA-HRP-reacted section. Most of the glomeruli in this section would have been categorized as fully stained. The number of glomeruli of each type was counted and data expressed as a percentage of total glomeruli in that section. For statistical analysis we combined the number of partially filled with fully filled glomeruli.
Turbinates were cut on a cryostat at 10 µm and directly thaw-mounted on chrome alum subbed slides. Sections were stained with cresyl violet combined with periodic acid Schiff or reacted for WGA-HRP with either direct DAB or nickel intensification. Standard DAB development of WGA-HRP used 0.01% H2O2 and 0.05% DAB (w/v) (Sigma) in 0.1 M phosphate-buffered saline.
Electron microscopy
Finally, two rats were prepared for electron microscopy after WGA-HRP injection. We perfused the rats with either 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate buffer or 1% paraformaldehyde and 1.25% glutaraldehyde in phosphate buffer. Sections were cut at 50 or 100 µm on a Vibratome and reacted with either DAB or DAB with nickel intensification. Intensely stained glomeruli were subsequently dissected from the coronal section, embedded in Epon and cut at 1 µm for light microscopic evaluation or at 90 nm (silver-gray) for electron microscopy. Sections were post-stained with lead citrate and uranyl acetate.
Statistical analysis
All statistical analyses were performed with SYSTAT9. Repeated measures analysis of variance (ANOVA) was used. Survival time following lesion (or no lesion) was the between-subjects measure and laminae or region (ventral, dorsal, medial or lateral) was the within-subjects measure. Post hoc analysis was performed with Bonferroni correction. In the event that a significant effect was found by ANOVA but the post hoc test did not detect a difference at the 0.05 level, the greatest mean difference was considered to be the only significant difference.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Olfactory bulb volumes following lesions
Figure 2 graphically presents laminar volumes calculated from the histological sections following olfactory nerve lesion. The overall F test (repeated measures ANOVA) showed no significant effect of days post-lesion on total bulb volume (F = 1.60, d.f. = 5,15, P = 0.22). Total volumes (means ± SE) ranged from 17.38 ± 1.26 mm3 in untreated rats to a minimum of 12.81 ± 1.45 mm3 at 9 days, with a return to essentially control values at 42 days (17.31 ± 1.26), however, variance among animals at each time point obscured any group significance.
|
|
A significant difference between laminar volumes was found, as would be expected (F = 523, d.f. = 4,60, P < 0.000). A significant lamina x post-lesion survival interaction showed that some laminae were affected differentially by lesion (F = 2.80, d.f. = 20,60, P < 0.001). Subsequent ANOVA found that the GL shrank significantly (F = 3.96, d.f. = 5,15, P < 0.02). Post hoc analysis disclosed a significant 45% shrinkage of the GL on day 9 compared with both the control and 42 days post-lesion. The EPL did not reach statistical significance (F = 1.83, d.f. = 5,15, P = 0.16). Neither the IPL nor the GCL showed significant changes (IPL: F = 0.48, d.f. = 5,15; P < 0.79; GCL: F = 0.65, d.f. = 5,15, P = 0.66). The ventricular layer (VENT) appeared to enlarge following lesion but did not reach statistical significance (F = 2.45, d.f. = 5,15, P = 0.08).
A correlational analysis disclosed substantial correlation among the variables and, in particular, a significant correlation between EPL volume and the combined volume of IPL, GCL and VENT (r = 0.90). These latter three areas showed no lesion effect (see above). Therefore, an analysis of covariance was performed on EPL volume with the combined volume of IPL, GCL and VENT as the covariate. This analysis showed that the EPL volume decreased following lesion when volume was adjusted for more central areas (F = 3.40, d.f. = 5,14, P < 0.03). Post hoc analysis did not find `significant' differences because of the conservative nature of Bonferroni contrasts. However, inspection showed a maximal decline of 26% at 9 days and a delayed recovery (Figure 3). Use of the same covariate did not change the statistical findings on GL shrinkage following ORN lesion. Conversely, use of the EPL as covariate eliminated the statistical significance of GL changes, which confirmed that EPL shrinkage was related to ONR lesion.
Glomerular areas
A factorial repeated measures ANOVA (with regional area as the repeated measure and time after lesion as the between-subjects factor) found both a significant main effect of time after lesion (F = 7.35, d.f. = 5,15, P < 0.001) and a significant regional effect (F = 34.37, d.f. = 3,45, P < 0.000). In addition, a significant region x time after lesion effect was found (F = 2.27, d.f. = 15,45, P < 0.02). Post hoc testing disclosed that dorsal glomeruli were smaller than glomeruli of other regions irrespective of lesion effects.
Equivalent lesion effects (i.e. shrinkage) occurred in all glomeruli (Figure 4) after ORN lesion. The interaction showed that glomerular area recovery was more rapid in ventral and lateral glomeruli than in medial and dorsal glomeruli. These differences were clearly obvious in Nissl stained sections (Figure 5). Post hoc testing of ventral glomeruli showed a 40-50% decline at day 9 from both the control and 42 day groups. Lateral glomeruli area differed significantly from the control and 42 days post-lesion groups at 3 and 9 days. Medial glomeruli area displayed a delayed recovery and was significantly different from the control and 42 day groups at both days 9 and 16. Finally, dorsal glomeruli were different from controls 3, 9 and 16 days following lesion. In sum, all glomeruli shrank equivalently following nerve lesion, but recovery was more rapid in ventral and lateral glomeruli than in medial and dorsal glomeruli.
|
|
WGA-HRP tracing in the olfactory bulb
We evaluated several concentrations and methods of application of WGA-HRP
described in previous reports. Highly variable labeling of glomeruli in
untreated rats occurred with concentrations of 1-2% WGA-HRP. Some glomeruli
stained well, but many displayed no observable labeling. Four percent WGA-HRP
improved but did not solve this problem. All quantification and most
observations were performed with 4% WGA-HRP and nickel-intensified reaction
product. We also found that occlusion of the contralateral naris during
WGA-HRP inspiration markedly reduced labeling. This suggests that decreasing
the inspiration rate of the naris ipsilateral to the irrigation is important
in obtaining more complete labeling, a conclusion implicit in a previous
report (Schwob et al.,
1999).
We obtained labeling of most glomeruli (90%) in untreated rats using a slow drip technique and 4% WGA-HRP (Figure 1). No regional selectivity for labeling was identified in control rats (Figure 6). Very occasionally we observed WGA-HRP-labeled neurons adjacent to intensely labeled glomeruli. In contrast to controls, weak labeling was not a problem in the rats following lesion. A diffuse, weak accumulation of reaction product throughout the glomerulus, as seen in some control rats, was not present in the lesioned rats until 42 days post-lesion, when reinnervation in most glomeruli tended to be complete (judging by area measures).
|
Effects of lesion and glomerular labeling
Figure 6 displays the combined percentage of glomeruli either fully or partially labeled following olfactory nerve lesion. Statistical testing showed significant variation in labeling with region (F = 13.67, d.f. = 3,45, P < 0.000) and with days post-lesion (F = 14.03, d.f. = 5,15, P < 0.000). The interaction region x time post-lesion failed to achieve an accepted level of significance (F = 1.80, d.f. = 15,45, P = 0.065). The main effect of regional variability represented delayed recovery by medial and dorsal glomeruli compared with ventral and lateral glomeruli. Post hoc testing within each region showed that WGA-HRP labeling of ventral glomeruli decreased only at 3 days (27% of ventral glomeruli showed some labeling). Labeling increased at 9 days, when almost 70% of ventral glomeruli showed either full or partial labeling (raw data 30, 90, 100%). WGA-HRP labeled significantly fewer lateral glomeruli at both 3 and 9 days than either the control group or on days 16-42 post-lesion. Medial glomeruli showed a lower labeled percentage at 3, 9 and 16 days than either the control or 42 days post-lesion group. Very high inter-animal variation masked differences in the dorsal glomeruli versus the controls except at 9 days, but the pattern was similar to medial glomeruli. The graphs of these data (Figure 6) show that WGA-HRP labeling of ventral and lateral glomeruli recovered more rapidly than did medial or dorsal glomerular labeling.
The subjective WGA-HRP activity largely paralled the morphometric analysis of glomerular size. At 3 days most glomeruli had little or no evidence of WGA-HRP labeling (Figure 7A). What little labeling was present in the bulb at 3 days was in the ventral or lateral glomeruli. At 9 days the ventral glomeruli in the middle third of the bulb showed labeling, but label was still absent in other regions (Figure 7B). Substantial differences appeared at 16 days and later. At 16 days most ventral and lateral glomeruli contained WGA-HRP in the middle two-thirds of the bulb (Figure 7C). Conversely, medial glomeruli were less intensely labeled at this stage (Figure 7D,E).
|
The pattern of labeled glomeruli at 16 days clarified the pattern of regeneration. Analysis of serial stained sections, although not quantified, showed that most glomeruli at or behind the level of the anterior olfactory nucleus displayed label (Figure 8A,B) and label was present two-thirds of the way through the OB (from the caudal-most glomeruli; Figure 8C,D). Sections rostral to this level displayed WGA-HRP in the nerve, but few glomeruli were labeled (Figure 8E,F). This reconstruction suggests that new fibers enter the denervated OB about one-third of the way from the rostral tip in the ventral layer and progress rostrally and caudally.
|
We observed little glomerular labeling contralateral to the injected naris.
This observation was not carefully explored, but our impression is that
relatively little solution infused in one naris was able to gain access to the
contralateral turbinates. These observations suggest that unilateral lesions
of the rat olfactory epithelium are possible
(Turner and Perez-Polo, 1993).
It is worth nothing that the same procedure in mice resulted in bilateral
labeling (Struble, personal observation).
Electron microscopy
Either fixative resulted in good visualization of the WGA-HRP product. In thick plastic-embedded sections (1 µm) the DAB product was more visible than the nickel intensification product. At the electron microscopic level the reaction product could be seen in processes that contained large numbers of vesicles (Figure 9). On this basis we conclude that they were olfactory afferents. It was also obvious that not all olfactory afferents in a single densely labeled glomerulus displayed reaction product.
|
Olfactory epithelium
We were generally unable to detect WGA-HRP reaction product in olfactory
epithelium (data not shown). When present, labeling was most common in the
lateral turbinates. ORN, sustentacular cells and, presumably, macrophages in
damaged areas of the olfactory epithelium displayed WGA-HRP activity. No
labeling was identified in the portions of the turbinates that surrounded the
central core of the olfactory turbinates. We attribute this lack of labeling
to xenobiotic activity, rapid clearance of WGA-HRP from the olfactory
epithelium or some aspect of tissue preparation that is incompatible with
maintaining WGA-HRP activity (Moon and
Baker, 1998).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Our studies extend previous reports of the effects of olfactory nerve lesion on the OB (Pinching and Powell, 1971; Margolis et al., 1974
; Harding et al.,
1977; Nadi et al.,
1981; Baker et al.,
1983; Stone et al.,
1991; Schwob et al.,
1995; Ravi et al.,
1997; Sashihara et
al., 1997; Setzer and
Slotnick, 1998; Schwob et
al., 1999; Weruaga et
al., 2000). Our volumetric analyses extend these studies and
show that the response of the OB can be basically broken into two
well-separated time periods. The initial response is degeneration, which
occurs until 7-8 days. Subsequently, regenerative responses appear and
continue until 6-8 weeks. Degeneration
Our data support both direct and transneuronal changes in the OB. Our
previous analysis of freshly obtained OB identified a statistically
significant 20% decrease at 3-7 days
(Struble et al.,
1999). Our current data show a statistically non-significant bulb
volume shrinkage of 26%. Non-significant changes in the histological analysis
is largely a variance problem. In our previous study
(Struble et al.,
1999) variance in the lesion group was quite small soon after
lesion (see Table 1) (Struble et
al., 1999). The olfactory nerve in the current study
sustained variable damage during dissection and histological processing and we
could not reliably quantify olfactory nerve. Basically, the variance among
rats in this study had increased relative to the variance under conditions
resulting in a non-significant effect. However, both the GL and EPL
shrank.
Nerve damage with DDTC clearly causes glomerular shrinkage. Individual
glomeruli shrink by 40% on day 3 and further decline to 50% of control at
9 days. This magnitude of individual glomerulus shrinkage at 3 days
corresponds to our estimate that, on average, 75% of the olfactory epithelium
is damaged by DDTC (Struble et
al., 1998) and that olfactory nerve terminals comprise
50% of the glomerulus (Hinds and
McNelly, 1981). In contrast to individual glomeruli, the GL shrink
by a non-significant 22% at 3 days and a statistically significant 45% by 9
days after lesion. The implication of the temporal difference in shrinkage of
glomeruli (3 days) versus the GL (9 days) is that the total glomerular layer
shrinkage represents both the 40-50% shrinkage of glomeruli and subsequent
shrinkage in the neuropil surrounding the glomeruli. This observation has
precedent in studies reporting tyrosine hydroxylase decline in periglomerular
neurons following nerve lesion (Nadi et al., 1974; Baker et
al., 1983,
1984; Margolis et al.,
1983). Dendritic and axonal fields of tyrosine hydroxylase-containing
periglomerular interneurons probably shrink and contribute to the total GL
shrinkage (Baker et al.,
1984). Similar changes could occur in other neurons and in
terminal dendritic fields of mitral and tufted neurons
(Pinching and Powell, 1972)
and account for the delayed EPL shrinkage discussed below.
The EPL follows the same temporal pattern as the GL, declining by only 6% on day 3 and 26% at day 9 after lesion. This difference is not statistically significant until we adjusted for volume by analysis of covariance with the subjacent laminae. Adjustment makes intuitive sense since the granule cells of the GCL provide much of the neuropil for the EPL. Use of EPL volume as a covariate for GL volume eliminates some inter-animal variability. However, it also eliminates the statistical significance of lesion on the GL. This result confirms that changes within the EPL are related to the same factor (nerve lesion) that caused GL shrinkage.
It is important to point out that laminar shrinkage occurs in the presence
of extensive reactive gliosis following nerve lesion
(Barber and Dahl, 1987;
Anders and Johnson, 1990;
Ravi et al., 1997;
Schwob et al., 1999).
Therefore, transneuronal changes may be more extensive than our data show. It
is also interesting that the ventricular layer shows non-significant evidence
of expansion several weeks after lesion and this difference became significant
(P = 0.02) when the EPL is used as a covariate. The biological
significance of this change may be related to functions of the subependymal
layer in possible gliagenesis and neurogenesis occurring after ORN lesion
(Peretto et al.,
1999)
The WGA-HRP tracing studies confirm and extend the volumetric aspects of
this study. We initially had problems in obtaining OB labeling in control rats
when using previously described techniques
(Shipley, 1985;
Stewart, 1985;
Baker and Spencer, 1986), but
these problems were usually ameliorated with a higher concentration of
WGA-HRP. Nonetheless, our electron microscopic data from control rats showed
unfilled olfactory terminals even when glomeruli were selected for intense
WGA-HRP activity. It is clear that although WGA-HRP was transported into the
OB, labeling with our procedure was incomplete. We had fewer problems with
weak labeling after lesion, presumably because either airflow or the
xenobiotic scavenging system was impaired in the lesioned rats. However,
several of the control rats and one lesioned rat used in the morphometric
studies showed no evidence of labeling even with 4% WGA-HRP, hence we had to
supplement this group in the tracing study.
WGA-HRP tracing confirmed previous estimates of damage by DDTC
(Struble et al.,
1998). Seventy to eighty percent of glomeruli are devoid of any
visible WGA-HRP reactivity 3 days following lesion. It is noteworthy that the
significant WGA-HRP labeling decline at 3 days compares well with glomerulus
shrinkage but not with GL shrinkage. This further supports transneuronal
changes underlying volumetric changes; loss of the nerve occurs by 3 days, but
shrinkage continued for another 6 days.
Regeneration
Volumetric recovery of the OB occurred between 9 and 16 days after
olfactory nerve lesion. Importantly, the morphometric estimates of glomerular
recovery and the WGA-HRP transport study are complementary. Approximately 70%
of ventral and lateral glomeruli are devoid of WGA-HRP activity at 3 days.
Regenerating fibers appear to return via the ventral OB surface and ventral
glomeruli show initial recovery of WGA-HRP labeling. At 9 days 73% of ventral,
but only 31% of lateral, glomeruli showed some label. By 16 days 100% of
ventral glomeruli and 86% of lateral glomeruli showed WGA-HRP. Medial
glomeruli labeling lagged both ventral and lateral labeling. Our data are
compatible with a previous study (Schwob
et al., 1999) that estimated that reinnervation occurred
during weeks 2-3. Their estimate is correct for total glomeruli, but the
ventral and some lateral glomeruli appear to be reinnervated earlier, i.e. at
9 days. These studies are also congruent with recovery studies in the
mouse (Verhaagen et al.,
1990). Any discrepancy between our data and data previously
published most likely relates to differential reinnervation of glomeruli
discussed below.
Obvious preferential reinnervation occurs when comparing both glomerular
area and WGA-HRP labeling. Laterally placed glomeruli in the middle portions
of the OB recover both area and WGA-HRP labeling before most medial glomeruli.
Medial and dorsal glomeruli in the rostral two-thirds of the OB appear to
recover much more slowly. This ventral-lateral-medial gradient is not an
artifact of the technique. Close inspection of photomicrographs in three other
reports shows a comparable gradient following either physical or chemical
lesion of the nerve (Yee and Costanzo,
1995; Setzer and Slotnick,
1998; Schwob et al.,
1999). Preferential sparing of ORN is not an attractive hypothesis
because different lesion techniques, which could selectively lesion epithelial
regions, should result in different patterns of sparing. However, the same
pattern of regeneration is seen with both chemical and mechanical lesions.
One possible explanation for a medial-lateral difference is the epithelial distribution of ORN cells projecting to glomeruli. The presence of very weak labeling of ventral and lateral glomeruli at 3 days presumably represents axons spared by the toxin, the number of which are sufficient to visualize WGA-HRP. It is possible that the ventral and lateral glomeruli may get a more dispersed projection from the epithelium than do medial and dorsal glomeruli. Lesion of epithelium, by any method, may result in sparing of some innervation and spared axons could act to guide growing axons to ventral and lateral glomeruli. It is also possible that the greater distance of dorsal and medial glomeruli from the olfactory epithelium may require more time for axons to arrive.
An alternative, although less simple, hypothesis is suggested by a previous
report of a protein preferentially expressed in ventral and lateral olfactory
epithelium (Schwob and Gottlieb,
1986) which project to the homologous region of the OB
(Astic and Saucier, 1986;
Saucier and Astic, 1986).
Subsequent studies proposed that this protein is a putative neural cell
adhesion molecule (Alenius and Bohm,
1997). Perhaps this protein facilitates growth of axons from these
regions. Why preferential reinnervation occurs is not clear, but differential
recovery permits within-animal use of the OB for systematic neuroanatomical
studies of central changes during reinnervation.
Recovery of WGA-HRP nerve labeling appears to parallel recovery of the
glomerular area except in ventral glomeruli, where a discrepancy may exist
between reinnervation, assessed with WGA-HRP, and glomerular area recovery. In
essence, WGA-HRP shows `complete' labeling of ventral glomeruli at 2 weeks,
although the size of ventral glomeruli had not recovered by 4 weeks (compare
Figures 4 and
6). Full recovery requires 6
weeks. Axonal growth in young animals has been described as `over-exuberant'
(Cummings et al.,
2000) and one study of reinnervation of identified axons suggested
axonal reorganization occurring after 10 weeks
(Costanzo, 2000). We suspect
that early arriving axons may initially enter ventral glomeruli (as shown with
WGA-HRP) but may not establish a functional relationship which would be
reflected by area recovery.
In sum, these studies present morphometric data on degeneration and regeneration of the OB subsequent to a reversible lesion of the olfactory epithelium. Both morphometric and tracing studies identify significant transneuronal changes, present a better time line of regeneration and identify a previously unrecognized gradient in OB reinnervation. These data further support the use of the OB as a unique experimental system for analysis of degeneration and regeneration-associated processes that occur in the central nervous system.
|
Acknowledgments |
|---|
We wish to acknowledge the work of Stephanie Perry on aspects of this project. This work was supported by NIH grants AG08014 (R.G.S.) and DC03889 (B.P.N.) and a Southern Illinois University Central Research Committee grant (R.G.S.), an Illinois Department of Public Health AD grant (B.P.N.) and Eastern Illinois University CFR grants (B.P.N.). Part of these data was previously presented in abstract form at the American Association of Neuropathologists
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Alenius, M. and Bohm, S. (1997) Identification of a novel neural cell adhesion molecule-related gene with a potential role in selective axonal projection. J. Biol. Chem., 272,26083 -26086.
Anders, J.J. and Johnson, J.A. (1990) Transection of the rat olfactory nerve increases glial fibrillary acidic protein immunoreactivity from the olfactory bulb to the piriform cortex.Glia , 3,17 -25.[ISI][Medline]
Astic, L. and Saucier, D. (1986) Anatomical mapping of the neuroepithelial projection to the olfactory bulb in the rat. Brain Res. Bull., 16,445 -454.[ISI][Medline]
Baker, H. and Spencer, R.F. (1986) Transneuronal transport of peroxidase-conjugated wheat germ agglutinin (WGA-HRP) from the olfactory epithelium to the brain of the adult rat.Exp. Brain Res. , 63,461 -473.[ISI][Medline]
Baker, H., Kawano, T., Margolis, F.L. and Joh, T.H. (1983) Transneuronal regulation of tyrosine hydroxylase expression in olfactory bulb of mouse and rat. J. Neurosci., 3,69 -78.[Abstract]
Baker, H., Kawano, T., Albert, V., Joh, T.H., Reis, D.J. and Margolis, F.L. (1984) Olfactory bulb dopamine neurons survive deafferentation-induced loss of tyrosine hydroxylase.Neuroscience , 11,605 -615.[ISI][Medline]
Barber, P.C. and Dahl, D. (1987) Glial fibrillary acidic protein (GFAP)-like immunoreactivity in normal and transected rat olfactory nerve. Exp. Brain Res.,65 , 681-685.[ISI][Medline]
Benzing, W.C. and Mufson, E.J. (1995) Apolipoprotein E immunoreactivity within neurofibrillary tangles: relationship to tau and PHF in Alzheimer's disease. Exp. Neurol., 132,162 -171.[ISI][Medline]
Costanzo, R.M. (2000) Rewiring the
olfactory bulb: changes in odor maps following recovery from nerve
transection. Chem. Senses, 25,199
-205.
Cotman, C.W., Hailer, N.P., Pfister, K.K., Soltesz, I. and Schachner, M. (1998) Cell adhesion molecules in neural plasticity and pathology: similar mechanisms, distinct organizations?Prog. Neurobiol. , 55,659 -669.[ISI][Medline]
Cummings, D.M., Emge, D.K., Small, S.L. and Margolis, F.L. (2000) Pattern of olfactory bulb innervation returns after recovery from reversible peripheral deafferentation. J. Comp. Neurol. 421,362 -373.[ISI][Medline]
Greer, C.A. (1991) Structural organization of the olfactory system. In Getchell, T.V., Doty, R.L., Bartoshuk, L.M., Snow, J.B., Pfaffmann, C. and Halpern, B.P. (eds), Smell and Taste in Health and Disease. Raven Press, New York, NY, pp.65 -81.
Harding, J., Graziadei, P.P.C., Monti Graziadei, G.A. and Margolis, F.L. (1977) Denervation in the primary olfactory pathway of mice. IV. Biochemical and morphological evidence for neuronal replacement following nerve section. Brain Res.,132 , 11-28.[ISI][Medline]
Hinds, J.W. and McNelly, N.A. (1981) Aging in the rat olfactory system: correlation of changes in the olfactory epithelium and olfactory bulb. J. Comp. Neurol.,203 , 441-453.[ISI][Medline]
Itaya, S.K. (1987) Anterograde transsynaptic transport of WGA-HRP in rat olfactory pathways.Brain Res. , 409,205 -214.[ISI][Medline]
Margolis, F.L., Roberts, N., Ferriero, D. and Feldman, J. (1974) Denervation in the primary olfactory pathway of mice: biochemical and morphological effects. Brain Res.,81 , 469-483.[ISI][Medline]
Moon, Y.W. and Baker, H. (1998) Induction of cell division in olfactory basal epithelium following intransal irrigation with wheat germ agglutinin-horseradish peroxidase.J. Comp. Neurol. , 393,472 -481.[ISI][Medline]
Nadi, N.S., Head, R., Grillo, M., Hempstead, J., Grannot-Reisfeld, N. and Margolis, F.L. (1981) Chemical deafferentation of the olfactory bulb: plasticity of the levels of tyrosine hydroxylase, dopamine and norepinephrine. Brain Res., 213,365 -377.[ISI][Medline]
Peretto, P., Merighi, A., Fasolo, A. and Bonfanti, L. (1999) The subependymal layer in rodents: a site of structural plasticity and cell migration in the adult mammalian brain.Brain Res. Bull. , 49,221 -43[ISI][Medline]
Pinching, A.J. and Powell, T.P.S. (1972)
Ultrastructural features of transneuronal cell degeneration in the
olfactory bulb. J. Cell Sci., 8,253
-287.
Rall, W., Shepherd, G.M., Reese, T.S. and Brightman, M.W. (1966) Dendrodendritic synaptic pathway for inhibition in the olfactory bulb. Exp. Neurol.,14 , 44-56.[ISI][Medline]
Ravi, R., Krall, A., Rybak, L.P. and Struble, R.G. (1997) Olfactory mucosal lesions following subcutaneous diethyl dithiocarbamate (DDTC). J. Neurotoxicol.,18 , 123-128.
Sashihara, S., Waxman, S.G. and Greer, C.A. (1997) Downregulation of Na+ channel mRNA in olfactory bulb tufted cells following deafferentation.Neuroreport. 8,1289 -1293.[ISI][Medline]
Saucier, D. and Astic, L. (1986) Analysis of the topographical organization of olfactory epithelium projection in the rat. Brain Res. Bull.,16 , 455-462.[ISI][Medline]
Schwob, J.E. and Gottlieb, D.I. (1986) The primary olfactory projection has two chemically distinct zones.J. Neurosci. , 6,3393 -3404.[Abstract]
Schwob, J.E., Youngentob, S.L. and Mezza, R.C. (1995) Reconstitution of the rat olfactory epithelium after methyl bromide-induced lesion. J. Comp. Neurol.,359 , 15-37.[ISI][Medline]
Schwob, J.E., Youngentob, S.L., Ring, G., Iwema, C.L. and Mezza, R.C. (1999) Reinnervation of the rat olfactory bulb after methyl bromide-induced lesion: timing and extent of reinnervation. J. Comp. Neurol.,412 , 439-457.[ISI][Medline]
Setzer, A.K. and Slotnick, B. (1998) Disruption of axonal transport from olfactory epithelium by 3-methylindole. Physiol. Behav.,65 , 479-487.[Medline]
Shipley, M.T. (1985) Transport of molecules from nose to brain: transneuronal anterograde and retrograde labeling in the rat olfactory system by wheat germ agglutinin-horseradish peroxidase applied to the nasal epithelium. Brain Res. Bull.,15 , 129-142.[ISI][Medline]
Stewart W.B. (1985) Labeling of olfactory bulb glomeruli following horseradish peroxidase lavage of the nasal cavity. Brain Res., 347,200 -203.[ISI][Medline]
Stone, D.M., Grillo, M., Margolis, F.L., Joh, T.H. and Baker, H. (1991) Differential effect of functional olfactory bulb deafferentation on tyrosine hydroxylase and glutamic acid decarboxylase messenger RNA levels in rodent juxtaglomerular neurons.J. Comp. Neurol. , 311,223 -233.[ISI][Medline]
Struble, R.G., Dhanraj, D.N., Mei, Y., Wilson, M., Wang, R. and Ramkumar, V. (1998) Beta-amyloid precursor protein-like immuno-reactivity is upregulated during olfactory nerve regeneration in adult rats. Brain Res.,780 , 129-137.[ISI][Medline]
Struble, R.G., Husain K. and Somani, S.M. (1999) Response of the olfactory bulb antioxidant system following diethyldithiocarbamate (DDTC) administration in rats. J. Appl. Toxicol., 19,221 -228[ISI][Medline]
Turner, C.P. and Perez-Polo, J.R. (1993) Expression of p75NGFR in the olfactory system following peripheral deafferentation. NeuroReport, 4,1023 -1026.[ISI][Medline]
Verhaagen, J., Oestreicher, A.B., Grillo, M., Khew-Doodall, Y.-S., Gispen, W.S. and Margolis, F.L. (1990) Neuroplasticity in the olfactory system: differential effects of central and peripheral lesions of the primary olfactory pathway on the expression of B-50/GAP-43 and the Olfactory Marker Protein. J Neurosci. Res., 26,31 -44.[ISI][Medline]
Weruaga, E., Brinon, J.G., Porteros, A., Arevalo, R., Aijon, J. and Alonso, J.R. (2000) Expression of neuronal nitric oxide synthase/NADPH-diaphorase during olfactory deafferentation and regeneration. Eur. J. Neurosci. 12,1177 -1193.[ISI][Medline]
Yee, K.K. and Costanzo, R.M. (1995) Restoration of olfactory mediated behavior after olfactory bulb deafferentation. Physiol. Behav.,58 , 959-968.[Medline]
Accepted May 1, 2001
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  E. Au, M. W. Richter, A. J. Vincent, W. Tetzlaff, R. Aebersold, E. H. Sage, and A. J. Roskams SPARC from Olfactory Ensheathing Cells Stimulates Schwann Cells to Promote Neurite Outgrowth and Enhances Spinal Cord Repair J. Neurosci., July 4, 2007; 27(27): 7208 - 7221. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||