Chronic neuroinflammation is thought to play an etiological role in Alzheimer’s disease (AD) which is characterized pathologically by amyloid and tau formation, as well as neuritic dystrophy and synaptic degeneration. The causal relationship between these pathological events is a topic of ongoing research and discussion. Recent data from transgenic AD models point to a tight spatio-temporal link between neuritic and amyloid pathology, with the obligatory enzyme for β-amyloid (Aβ) production, namely β-secretase-1 (BACE1), being overexpressed in axon terminals undergoing dystrophic change. However, the axonal pathology inherent with BACE1 elevation seen in transgenic AD mice may be secondary to increased soluble Aβ in these genetically modified animals. Further, it is unclear whether the inflammation seen in AD is the result of , or the cause of neuritic dystrophy. Here we explored the occurrence of AD-like axonal and dendritic pathology in adult rat brains affected by LPS-induced chronic neuroinflammation. Unilateral intracerebral LPS injection induced prominent inflammatory response in glial cells in the ipsilateral cortex and hippocampal formation. BACE1 protein levels were elevated in the ipsilateral hippocampal lysates in the LPS-treated animals relative to controls. BACE1 immunoreactive dystrophic axons appeared in the LPS-treated ipsilateral cortex and hippocampal formation, colocalizing with increased β-amyloid precursor protein and Aβ antibody (4G8) immunolabeling. Quantitative Golgi studies revealed reduction of dendritic branching points and spine density on cortical layer III and hippocampal CA3 pyramidal neurons in the LPS-treated ipsilateral cerebrum. These findings suggest that Alzheimer-like amyloidogenic axonal pathology and dendritic degeneration occur in wildtype mammalian brain in partnership with neuroinflammation following LPS injection.
Neuroinflammation has been linked to many neuropsychiatric disorders, including neurodegenerative diseases such as Alzheimer’s diseases (AD), Parkinson’s disease (PD), multiple sclerosis and traumatic brain injury [
Loss of synapses and their connectivity best correlate with cognitive deficits in AD [
The bacterial endotoxin lipopolysaccharide (LPS) can induce chronic neuroinflammation in rodents [
In-house bred male adult Sprague-Dawley rats (n = 24) weighing ~200 g (Animal Center of Central South University) were used. Rats were maintained in temperature (20˚C - 26˚C), humidity (30% - 60%) and lighting (12:12 hours light/dark cycle) controlled rooms, with food and water freely available. For intracerebral injections, rats were placed in a stereotaxic apparatus under sodium pentobarbital anesthesia (50 mg/kg, i.p.). LPS from Escherichia coli serotype 055:B5 (L2637, Sigma-Aldrich, St. Louis, MO, USA) was dissolved (2.5 µg/µl) in sterile phosphate-buffered saline (PBS, 0.01 M, pH 7.2) (vehicle) [
Hippocampi ipsilateral to the injection side were dissected out following decapitation (n = 4/group). Tissue was weighed and homogenized in a commercial protein extraction buffer at 1 to 10 w/v ratio (CW0883, Kangwei Century Company, Beijing), followed by centrifuge at 15,000 g. Protein concentrations in the supernatants were determined by DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). A total of 25 µg protein from each sample was separated electrophoretically in 10% SDS-polyacrylamide gel and transferred to polyvinylidene fluoride membrane. Membranes were blocked with 1% non-fat milk and 5% bovine serum albumin (BSA) in 0.1 M Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 2 hours at room temperature. Membranes were then incubated overnight at 4˚C in the same buffer containing rabbit anti-BACE1 (1:2000) [
Rats were perfused transcardially with PBS followed by 4% phosphate-buffered (0.1M) paraformaldehyde (pH 7.4). Brains were removed and postfixed for 12 hours and transferred into 30% sucrose for cryoprotection. Thirty micrometer-thick coronal sections were cut in a cryostat, with 12 sets of sections collected for cresyl violet stain and for immunohistochemical studies. For immunohistochemistry with the avidin-biotin complex (ABC) method, free-floating sections were soaked in 1% H2O2 in PBS for 30 minutes to diminish endogenous peroxidase activity, and preincubated in PBS buffer containing 0.2% Triton X-100 and normal horse serum for 1 hour. The sections were then reacted with one of the following primary antibodies: 1) Mouse monoclonal antibody against major histocompatibility complex class II molecules (MHC-II) at 1:1000 (ab55152, Abcam, Cambridge, MA, USA); 2) Rabbit anti-human BACE1 at 1:2000; 3) mouse anti-β-amyloid precursor protein (APP) monoclonal antibody 22C11 (MAB348, EMD Millipore, Billerica, MA, USA, 1:4000); 4) mouse anti-Aβ17-24 monoclonal antibody 4G8 (#39240, Signet, Dedham, USA, 1:4000); 5) rabbit anti-GFAP (G9269, Sigma-Aldrich, 1:2000). The sections were further reacted with a biotinylated pan-specific secondary antibody (BA-1300, Vector Laboratories, Burlingame, CA, USA, 1:400) for 2 hours, and subsequently with the ABC reagents (PK-6100, Vector Laboratories, 1:400) for one hour. Immunoreactivity was visualized in 0.05% 3,3’-diaminobenzidine (DAB) and 0.003% H2O2.
Selected sections were processed for double immunofluorescence beginning with blocking nonspecific reactivity by incubation in PBS buffer containing 5% normal donkey serum. Sections were further reacted overnight at 4˚C with a pair of primary antibodies raised in different species in PBS containing 0.2% Triton X-100 and the blocking serum. The antibody pairs included: 1) mouse anti-MCH-II (1:1000) and rabbit anti-CD11b (MABT149, EMD Millipore, 1:1000); 2) mouse anti-MCH-II and rabbit anti-GFAP (G9269, 1:2000); 3) rabbit anti-BACE1 and mouse anti-APP 22C11; 4) rabbit anti-BACE1 and mouse anti-Aβ 4G8; 5) rabbit anti-BACE1 and mouse anti-synaptophysin (MAB329, EMD Millipore, 1:4000); 6) rabbit anti-BACE1 and mouse anti-microtubule associated protein-2 (MAP2) (M9942, Sigma-Aldrich, 1:2000). On the second day, the sections were rinsed with PBS and incubated at room temperature for 2 hours with Alexa Fluor® 488 and Alexa Fluor® 594 conjugated donkey anti-mouse and anti-rabbit IgGs (1:200, Invitrogen, Carlsbad, CA, USA). Sections were then counter-stained with bisbenzimide (Hoechst 33342, 1:50000, Catalog #B2261, Sigma-Aldrich), washed thoroughly, and mounted with anti-fading medium before microscopic examination.
Brains were removed following a vascular rinse with PBS. Blocks containing the middle 1/3 cerebrum of both hemispheres were rinsed briefly in double distilled water, and processed with the FD Rapid Golgi stain TM Kit (FD Neuro Technologies, Ellicott City, MD) following the manufacturer’s instruction. The brain blocks were immersed in freshly made mixture of Solutions A and B (1:1) in the dark at room temperature for 2 weeks, and then in Solution C at 4˚C in the dark for 3 days. After silver impregnation, the blocks were cut slowly into frontal sections at 100 μm thickness in a vibratome. Sections were collected alternatively in 10 sets in Solution C, mounted on gelatin-coated microslides, dehydrated through ascending concentrations of ethanol, cleared in xylene, and sealed with PermountTM mounting medium.
An Olympus (BX53) microscope equipped with imaging system (CellSens Standard, Olympus) was used for examining sections stained with the ABC and fluorescent methods. Double immunofluorescence was also imaged on a confocal microscope (Nikon, DIGITAL ECLIPSE C1 plus, 5 μm thickness scan). Immunolabeling in sections around the level of injection in the rostrocaudal dimension was comparatively examined between the ipsilateral and contralateral cerebral hemispheres, using the needle track as a reference. Golgi-impregnated sections were examined on a Zeiss Axioplan microscope equipped with the Neurolucida and a high-resolution motorized stage for 3D neuronal reconstruction (MicroBrightField China). Two sections nearest to the injection coordinates (in the rostrocaudal dimension) were selected from each brain for neuronal morphometric analysis. Golgi-stained pyramidal neurons that met the following criteria were selected for reconstruction: 1) they were located in layer III of the parietal cortex overlying the mid-hippocampus and in the middle portion of CA3 (i.e., around the dorsal to ventral turning area); 2) they were among the labeled cells with the widest dendritic field by overall visual judgment; 3) they were well separated from other impregnated cells such that their dendritic tree was not or minimally overlapped with the processes from other cells; 4) the somata and dendritic processes were well-impregnated throughout the section thickness, with no apparent truncation of the dendritic arbor. Ten pyramidal neurons per region/brain were reconstructed with the aid of the Neurolucida software. Subsequently, somal area, total length of the dendritic tree, branching nodes and spine density (per 10 μm length) of the apical and basal dendrites, were obtained from a given selected neuron.
Imaging and numerical data for comparing groups were processed, with the mean ± SD calculated. Means were statistically analyzed by Student-t test or one-way ANOVA with posthoc Duncan’s multi-group comparisons when applicable. P < 0.05 was considered statistically significant. Figures were assembled with Photoshop 7.1, with brightness and contract adjusted as needed.
To confirm the occurrence of chronic neuroinflammation, cerebral sections from the LPS and PBS groups were processed under identical conditions for the detection of immunoinflammatory proteins. Compared to PBS controls, increased immunoreactivity for MCH-II (Figures 1(A)-(E)), CD11b (not shown) and GFAP (not shown) occurred in the ipsilateral cortex and hippocampal formation in the LPS-injected brain sections. Specifically, MCH-II immunoreactive cells appeared to be largely glial cells (
BACE1 protein levels were immunoblotted with a well-characterized rabbit antibody, which detects mature BACE1 protein migrating at ~70 kd [
Representative images illustrating immunoinflammatory cellular/molecular changes in rat cortex and hippocampal formation following intracerebral lipolysaccharide (LPS) injection. Panel (A) shows minimal immunoreactivity of major histocompatibility complex class II molecules (MHC II) in the hippocampal CA1 to CA3 areas, the dentate gyrus (DG) and the overlaying cortex (Ctx) in a PBS-injected control rat, with the area of the dentate gyrus enlarged as (C). MHC II immunolabeling is increased in both the cortex and hippocampal formation in the LPS-injected animal (B), with the labeled cellular profiles appeared as glial cells at high magnifications ((D), (E)). Confocal double immunofluorescence shows a great extent of colocalization of MHC II reactivity among CD11b labeled microglial cells in the LPS-treated cerebrum ((F)-(H)). A small amount of MHC II labeled cells exhibit glial fibrillary acidic protein (GFAP) immunoreactivity, suggestive of a colocalization in activated astrocytes ((I)-(K)). Scale bar = 500 μm in A applying to B; equivalent to 250 μm for ((C), (D)); and to 75 μm for ((E)-(K))
lotted BACE1 signal was significantly increased relative to PBS-treated counterparts (
In normal mammalian brains BACE1 immunoreactivity is largely expressed in the neuropil in a diffuse pattern
Western blot analysis showing elevation of the levels of β-secretase-1 (BACE1) and GFAP in the LPS-injected relative to PBS-treated (control) brains (n = 4/group), as assayed using hippocampal lysates ipsilateral to the injection. BACE1 levels in the LPS-treated group are about 2 times of that in the control ((A), (B)), while GFAP levels in the LPS group approach to 1.5 times of the control ((A), (C)), with the changes being significant for both proteins (paired two-tail t-tests)
except for a distinct heavy labeling at selected neuronal terminal fields including the mossy fiber terminals and olfactory glomeruli. In human and transgenic animal brains with amyloid plaque pathology, BACE1 immunoreactivity is increased preferentially in swollen and sprouting axonal terminals or axonal dystrophic neurites [
In double immunofluorescence, the BACE1 immunoreactive swollen neurites exhibited a great extent of colocalization with APP (Figures 4(A)-(H)). In addition, the Aβ antibody 4G8 (which detects mouse APP, Aβ and likely APP β-C-terminal fragments, ref. 34) showed increased immunoreactivity in the same area with BACE1 labeled neurites (Figures 4(I)-(K)). A colocalization of BACE1 and 4G8 labeling was detected among some individual neuritic profiles. Notably, there existed a considerable amount of diffuse extracellular Aβ labeling (not colocalized with BACE1 reactivity), some of which appeared punctate (
Representative images illustrating the occurrence of BACE1 immunoreactive neuritic pathology after focal LPS injection in rat cerebrum. In the vehicle control (injection of phosphate-buffered saline, PBS), the pattern of BACE1 immunoreactivity is comparable between the ipsilateral and contralateral cortex and hippocampal formation, which is generally associated with neuropil except for a heavy labeling at the hippocampal mossy fiber (mf) terminals (A). In the LPS injected animal (B), BACE1 immunoreactivity is increased in the neuropil in the ipsilateral relative to contralateral cortex and hippocampal formation, especially apparent around the needle track (outline by arrows). At high magnification, swollen neurites are found in the cortex and hippocampal formation in the ipsilateral side, especially evident in the white matter, stratum radiatum (s.r.), stratum-lacuno- sum-moleculare (s.l.m.) and the striatum oriens (s.o.) that show little labeling in the normal conditions ((C), (D), (F)). Panels ((E), (F)) are taken from the section that is ~480 μm (12 × 30 μm) apart from (B), showing the distinct difference regarding the neuritic pathology between the two sides. The needle entry at the cortical surface is marked with an asterisk (*). Ctx: cortex (grey matter); DG: dentate gyrus; PC: parietal cortex; s.l.m.: stratum-lacu- nosum-moleculare; s.p.: stratum pyramidale. WM: white matter; Scale bar = 1 mm in (A) applying to (B); equivalent for 400 μm for ((C), (D)) and 200 μm for (E)
sphericles and swollen processes were partially colocalized with the presynaptic marker synaptophysin (Figures 4(M)-(P)). In contrast, the BACE1 labeled swollen neurites did not colocalize with MAP2, which is clearly expressed in dendrites and somata of nearby pyramidal neurons (Figures 4(R)-(U)).
In our Golgi preparations, pyramidal neurons around layer III were consistently impregnated in all brain samples (
Confocal double immunofluorescent images showing LPS induced axonal pathology associated with increased labeling of amyloidogenic proteins. All images are taken from the CA3 area of the hippocampus ipsilateral to the intracerebral injection of PBS ((A)-(C)) or LPS ((E)-(U)). Antibody markers and color channels are as indicated. Panels ((A)-(C)) show double labeling of BACE1 and β-amyloid precursor protein (APP) in ipsilateral CA3 of the control animal, with the former expressed predominantly in the mossy fiber (mf) terminals, and the latter largely in somata of CA3 pyramidal neurons. Note that no abnormal neurites are present in the strum oriens (s.o.). Panels ((E)-(H)) illustrate the occurrence of BACE1/APP double labeled swollen neurites (examples are indicated by arrows) in the s.o. of the ipsilateral hippocampus of the LPS injected rat. Panels (I-L) show that these BACE1 positive neurites are locally associated with increased 4G8 labeling within (pointed by arrows) as well as outside (arrowheads) the swollen terminals in the cortex and CA1. Panels ((M)-(P)) show that a partial coexpression of synaptophysin (SYN) among the BACE1 labeled swollen neurites in the s.o. of the LPS injected ipsilateral hippocampus, which appear in yellow in the merged image (arrows, P). Panels (R-U) show that there is no colocalization of the microtubule associated protein-2 (MAP2) in the BACE1 labeled swollen neurites. MAP2 labeling is distinctly associated with the somata and dendrites of pyramidal neurons in the stratum pyramidale (s.p.). DAPI counterstain is included in panels (L) and (U), showing that the BACE1 labeled elements are not somatic. Scale bar = 200 μm in (A) applying to ((B), (C), (E)-(G) and (R)-(T)), equivalent to 100 μm for (I)-(K), (M)-(O)), 50 μm for ((D), (H), (U)) and 25 μm for ((L), (P))
CA1 direction and turns ventrally toward the dentate gyrus, could be defined systematically across brains. Therefore, we decided to use Golgi-stained pyramidal neurons in cortical layer III and around the dorsoventral turning area of CA3 for automated Neurolucida reconstruction and morphometry. Measurements from reconstructed layer III and CA1 pyramidal neurons in the ipsilateral hemisphere of the LPS and PBS treated brains
Quantitative Golgi study of somal and dendritic changes in cortical layer III pyramidal neurons in LPS-injected relative to PBS-injected rats. Panel (A) shows an example of Golgi stain in the parietal neocortex (ipsilateral to PBS injection), note that layer III pyramidal neurons are relatively well-labeled. Panels ((B), (C)) show representative Neurolucida reconstructions of impregnated layer III pyramidal neurons from a LPS (B) and a PBS (C) injected brains, with their apical and basal dendrites and dendritic branching points illustrated in color. Bar graphs ((D)-(G)) summarize the parameters obtained from 10 constructed neurons in each brain (n = 4/group). The means of somal perimeter and somal area show a trend of reduction in the LPS relative to PBS (control) groups, although the difference is not statistically significant (E). The means of total dendritic length tends to reduce in the LPS group relative to control, with significant difference for that of the basal dendritic tree (F). The branching nodes (points) are reduced in the LPS group on the basal dendritic tree (G). Overall spine density is significantly reduced on the dendritic tree of Golgi-impregnated cortical pyramidal neurons in the LPS group relative to control (H). Scale bar = 200 μm in (A)
(10 cells per region per brain) were compared quantitatively (Figures 5(A)-(C) and Figures 6(A)-(D)).
The somal perimeters of the layer III pyramidal neurons were comparable between the LPS (58.6 ± 7.4 µm, mean ± S.D., same format below) and PBS (61.7 ± 9.9 µm) groups (P > 0.05, two-tail paired t-test; same statistical test below) (
Quantitative Golgi study of somal and dendritic changes in in CA3 pyramidal neurons in LPS-injected relative to PBS-injected rat brains. Panel (A) shows an example of Golgi-stained CA3 pyramidal neurons located around dorsal to ventral turning area of the stratum pyramidale (s.r.). Basal dendrites in the stratum oriens (s.o.) and apical dendrites in the stratum radiatum (s.r.) are visible, with dendritic spines clearly labeled at high resolution (B). Panels ((C), (D)) show examples of Neurolucida reconstructions of CA3 pyramidal neurons from LPS (C) and a PBS (D) injected brains. The reduction in mean somal area (SA) in the LPS relative to PBS groups is statistically significant, whereas the reduction in the somal perimeter (SP) is not (E). The reduction in the total dendritic length in the LPS relative to PBS groups is significant for both the basal and apical trees (F). Scale bar = 50 μm in (A) and 5 μm for (B)
and 10.6 ± 2.7 for the two groups on the basal dendrites. Statistical tests reported a significant difference between the two groups for the means of the basal dendritic but not the apical measurements (
For the reconstructed CA3 pyramidal neurons (Figures 6(A)-(D)), both the somal perimeter and somal area of the pyramidal neurons tended to reduce in the LPS relative to PBS groups (
The LPS model is increasingly used in mechanistic and translational studies into chronic neurodegenerative dis
eases such as AD and PD [
We have shown in a recent study that unilateral intracerebral injection of ~10 μg LPS is sufficient to induce spatial learning and memory impairments in adult rats. Upregulation of immunoinflammatory molecules including PirB and GFAP is evident in the LPS-treated brains, occurring largely in the ipsilateral cortex and hippocampal formation [
Besides glial responses, neuronal and synaptic alterations have been reported in rodent LPS models anatomically and electrophysiologically. Local LPS infusion causes increased cell/neuronal death in the entorhinal cortex [
Here we demonstrate pre- and post-synaptic alterations among cortical and hippocampal neurons in LPS relative to PBS injected rats. Microscopically evident axonal pathology is featured by aberrant sprouting and swelling, and increased expression of APP, BACE1 and synaptophysin, but not MAP2. These abnormal axonal profiles seen following LPS treatment resemble the dystrophic neurites seen in models of injury that exhibit an increase in APP or A-beta [
LPS administration has been shown to influence amyloid pathogenesis via mixed cellular modulations in wild-type and transgenic rodents. Chronic intraventricular LPS infusion in rats induces intraneuronal Aβ immunoreactivity in the hippocampus [
Being the obligatory enzyme for Aβ genesis, BACE1 is at the crossing point potentially linking cellular and signaling substrates to amyloid pathogenesis. BACE1 elevation is associated with axonal pathology during plaque development in the brains of transgenic AD models as well as aged and AD human subjects [
More basic questions can be asked as to why axonal sprouting/dystrophy, dendritic/spine degeneration and glial activation develop in partnership under neuroinflammatory conditions. One possibility is that the toxin interrupts axonal transport causing accumulation of the amyloidogenic proteins and other neuronal molecules [
In summary, the present study demonstrates that chronic neuroinflammation induced by intracerebral LPS injection promotes the amyloidogenic cascade in dystrophic neurites by upregulation of BACE1 and APP in the brain. This modulation is evident in axon terminals that exhibit dystrophic-like morphology. LPS injection also induces degenerative changes in postsynaptic components manifested as dendritic shrinkage and spine loss among cortical and hippocampal pyramidal neurons. Together with the immunoinflammatory responses of the glial cells, the LPS model recapitulates multiple cellular and molecular deficits seen in aging and AD brains. The synaptic triad could be the initial site as well as center of neural degeneration and aberrant regeneration, with its progression ultimately leading to overt neuritic amyloid pathology and cognitive deficits.
This study was supported by the National Natural Science Foundation of China (#81171091, #81171160), Natural Science Foundation of Hunan Province (13JJ5016), Central South University (CSUZC2014035), Center for Alzheimer’s Disease and Related Disorders (P.R.P), and the Intramural research program of National Institute on Aging, NIH (Z01-IAAG000944-04 to H.C.).