Distinctive Toll-like Receptors Gene Expression And Glial Response in Different Brain Regions Of Natural Scrapie Part 2

2.2. Neuropathological Features in the Hippocampus of Sheep Naturally Infected with Serapie

In the scrapie-infected and control sheep, the four following hippocampal areas were examined in greater detail for all pathological markers: the pyramidal cell layers CA1, CA3, and CA4, and the SLM (Figure 3).

Recent studies have shown that the pyramidal cell layer plays an important role in memory. The pyramidal cell layer is neuronal in the brain mainly responsible for the perception of the external environment and memory storage.

During learning and memory, the pyramidal cell layer releases some neurotransmitters, which can stimulate communication between neurons, thereby enhancing the storage and retrieval of memory. In addition, the pyramidal cell layer can also screen and integrate information in the learning and memory process, helping us to quickly master new knowledge and skills.

Studies have also found that the function and health of the pyramidal cell layer have a great impact on the performance of memory. If the pyramidal cell layer is damaged or severely stressed, memory may be greatly affected. Therefore, we need to pay attention to the health and maintenance of the pyramidal cell layer to maintain good learning and memory abilities.

Recommendations for the health and maintenance of the pyramidal cell layer include diet, exercise, and sleep. For example, a diet rich in fatty acids and antioxidants can help promote the health of the pyramidal cell layer; regular exercise can enhance the function and resilience of the nervous system; and good sleep habits can help reduce the stress on the pyramidal cell layer and promote memory consolidation and retrieval.

In short, the pyramidal cell layer plays an important role in learning and memory. We should pay attention to the health and maintenance of the pyramidal cell layer to improve our learning and memory abilities. 

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These effects can help improve memory, learning ability, and thinking speed, and can also prevent the occurrence of cognitive dysfunction and neurodegenerative diseases.

Figure 3. Distribution of neuropathological lesions (HE), PrPSc deposition, astrogliosis (GFAP), and microgliosis (Iba1), in four different hippocampal regions in scrapie-infected and control sheep: pyramidal cell layers CornuAmmonis (CA) 1, 3, and 4, and the stratum lacunosum-moleculare (SLM). 

Graphs depict semi-quantitative assessment values for each parameter analyzed. (A) Mild spongiform changes were observed in all hippocampal regions analyzed. (B) Intraneuronal PrPSc pattern (L42) in CA1, CA3, and CA4 and widespread fine granular PrPSc pattern in the SLM. (C) Increased Iba1 staining in scrapie-infected versus control sheep. 

Magnified images show some of the morphological differences observed. (D) Signs of moderate reactive astrogliosis in scrapie-infected sheep compared with controls. Scores range from 0 (negative) to 5 (maximum intensity). 

Significant differences were determined using the Student's t-test or Mann–Whitney U test for normally and non-normally distributed data, respectively. * p < 0.05, ** p < 0.01. Scale bar = 200 µm.

Spongiosis in the hippocampus was very mild, and occasional vacuoles were observed in the CA1, CA3, and CA4, and the SLM (Figure 3A). This limited neuronal loss contrasted with sparse, but focally intense PrPSc deposition in CA1 and CA3 (Figure 3B). 

Very mild PrPSc staining was observed in the CA4 and the SLM. Distinct PrPSc patterns were observed in these brain areas (Figure S2). Whereas intraneuronal and neuropil-associated PrPSc patterns predominated in the CA1 and CA3, intraglial deposition was the main morphological pattern observed in the SLM. 

Mild intraneuronal and neuropil-associated PrPSc patterns were consistently observed in the CA4, although the intensity was lower than that observed in CA1 and CA3. Iba1 was constitutively expressed and sparsely distributed in the control samples. By contrast, marked microgliosis was observed in CA1, CA3, and CA4 and the SLM in scrapie-infected sheep. 

However, the microglial morphological features differed significantly between these brain regions. In the CA1 pyramidal layer, most microglial cells showed short processes with enlarged cell bodies, characteristic of the hypertrophic morphology. 

In CA4, the microglial cells were also hypertrophic but displayed longer and thinner branches than the CA1 microglial cells. Meanwhile, the hypertrophic microglia in the SLM showed short and thin processes with swollen cellular bodies. By contrast, in CA3, we observed the aforementioned rod-microglia morphology [42]. 

These microglial cells had elongated cell bodies, with a long parallel primary process oriented perpendicular to the CA3 pyramidal cell layer, and subsidiary branches that were shorter than the long primary processes. 

GFAP immunostaining was significantly increased in the CA1 and CA3 pyramidal cell layers from the scrapie-infected versus control sheep. Interestingly, the PrPSc scores were highest in CA1 and CA3. No differences were observed in the CA4 pyramidal cell layer or the SLM.

2.3. Neuropathology of Scrapie-Infected tg338 Mice

The same neuropathological markers of spongiosis, PrPSc deposition, microgliosis, and astrogliosis analyzed in sheep were examined in the thalamus of clinically affected tg338 mice that had been intracerebrally infected with scrapie (Figure 4). 

Compared with the controls, significant differences (p < 0.05) in spongiosis were observed in the thalamus of scrapie-infected mice, in which the vacuoles were restricted to the neuropil. 

PrPSc deposits were widely distributed at the level of the cytoplasm and neuropil. The assessment of gliosis revealed significant differences in Iba1 and GFAP immunostaining between the scrapie-infected and control mice, with both astrocytes and microglia displaying hypertrophic morphologies. 

The PrPres accumulation was also assessed by WB in all scrapie-infected mice, revealing a constant 19 kDa band pattern (Figure 5). The intensity of the PrPres signal in tg338 mice was more homogeneous among individuals than in sheep, as expected for experimental infection in which the dose, age of inoculation, and time of sacrifice were controlled. 

No signal was detected in the control mice inoculated with normal brain homogenate, in agreement with the negative IHC results.

Figure 4. Thalamus sections from scrapie-infected (intracerebral inoculation) and age-matched tg338 control mice immunostained to assess spongiosis (hematoxylin-eosin), PrPSc deposition (R486 antibody), astrocytes (glial fibrillary acidic protein; GFAP), and microglia (ionized calcium-binding adaptor molecule-1; Iba1). (A, E) Severe spongiform lesions located mainly in the neuropil of scrapie-infected versus control mice. (B, F) Several PrPSc-positive neurons are evident in scrapie-infected mice, but absent from controls. (C, G) Activated microglia displaying more intense immunostaining and thick processes in scrapie-infected mice versus controls. (D, H) Increased astrogliosis with hypertrophic cell bodies and processes in scrapie-infected mice versus controls. Scores range from 0 (negative) to 5 (maximum intensity). 

Significant differences were determined using the Student's t-test or Mann–Whitney U test for normally and non-normally distributed data, respectively. * p < 0.05, ** p < 0.01. Scale bar = 200 µm.

Figure 5. Western blot detection of PrPres accumulation in the CNS of clinically affected tg338 mice intracerebrally inoculated with scrapie compared with matched controls. 

Equivalent protein quantities were loaded for adequate comparison and immunoblots were run using SHA31 monoclonal antibody. Molecular-weight markers (kDa) are indicated on the left side of the immunoblot.

The upregulation of TLR expression in response to Prpsc has been previously reported in experimental in vivo and in vitro conditions, Therefore, after characterizing the presence of the main prion-induced neuropathological changes in the CNS in the context of natural and experimental disease, we investigated whether these changes correlated with alterations in TLR gene expression. 

To this end, we performed qPCR using samples from the four brain regions of sheep to assess the expression of MyD8g and Trif, the two main adaptor proteins implicated in TLR signaling; CD36, a scavenger receptor that cooperates with TLRs; and TLR mRNA. 

In addition, we measured the levels of four pro- and anti-. inflammatory cytokines in the thalamus and hippocampus of sheep to further assess the inflammatory response. In tg338 mice, we measured the mRNA levels of MyD88 and TLR1-9 in the thalamus. 

Genes for which significant differences were observed concerning controls, and the corresponding fold changes, are shown in Table $l, and the ACt values from the control and scrapie-infected sheep are represented in Figure $3.

The number of altered genes decreased in a caudal-rostral direction. The medulla oblongata of scrapie-infected sheep was the region in which the greatest changes in TLR gene expression were observed (Figure 6). In this region, the expression of TLR2, 3, 4, 6, 7, 8, 9, and CD36 was significantly higher (p < 0.01) in the scrapie-infected versus control animals.
In the thalamus, TLR6, MyD88, and CD36 were significantly upregulated in the scrapie-infected group (p < 0.05), while TLR2 and TLR3 displayed a tendency towards upregulation (p < 0.1). In the frontal cortex (the rostralmost area), TLR4 and CD36 were the only overexpressed genes (p < 0.05). 

Remarkably, contrasting expression patterns were found in the hippocampus of the scrapie-infected sheep, in which TLR2 and MyD88 were downregulated (p < 0.01) and TLR1 showed a tendency towards downregulation (p < 0.1). 

In addition, the hippocampus was the only area in which CD36 expression was not increased. Overall, TLR4 was the gene that was most upregulated in scrapie-infected versus control sheep in all four brain regions, although no significant differences were observed in the thalamus or hippocampus due to intra-individual variability. 

The analysis of cytokine levels revealed the overexpression of TGF-β, IL-10, and IL-6 in the thalamus of the scrapie-infected versus control sheep, but no alterations in the hippocampus (Figure 7).

Figure 6. Gene expression of TLR 1-10, MyD88, Trif, and CD36 in the medulla oblongata (A), thalamus (B), frontal cortex (C), and hippocampus (D) of control and scrapie-infected sheep. 

Data are represented as the mean fold ± SEM increase concerning control sheep (expressed as a score of 1). Mean scores were compared using the Student's t-test or Mann–Whitney U test for normally and non-normally distributed data, respectively. # p < 0.1, * p < 0.05, ** p < 0.01.

Figure 7. Gene expression of the anti-inflammatory cytokines TGF-β and IL-10, and the proinflammatory cytokines TNF-α and IL-6, in the thalamus (A) and hippocampus (B) of control and naturally scrapie-infected sheep. 

Data are represented as the mean ± SEM fold change concerning controls (expressed as a score of 1). Mean scores were compared using the Student's t-test or Mann–Whitney U test for normally and non-normally distributed data, respectively. * p < 0.

To examine whether changes in TLR expression in the CNS in natural disease are comparable to those induced in the tg338 murine model after intracerebral infection with scrapie, the transcription levels of TLRs and MyD88 were evaluated in the thalamus of tg338 mice (Figure 8). TLR1 and TLR2 mRNA levels were significantly increased (p < 0.01) compared with controls, and there was a tendency towards upregulation of TLR7 (p < 0.1). The levels of TLR8 expression were undetected.

Figure 8. Gene expression of TLR1-7, TLR9, and MyD88 in the thalamus of clinically affected tg338 mice intracerebrally inoculated with scrapie-positive inoculum versus controls (scrapie-negative inoculum). Results are expressed as the mean ± SEM fold change concerning controls (expressed as a score of 1). Mean scores were compared using the Student's t-test or Mann–Whitney U test for normally and non-normally distributed data, respectively. # p < 0.1, ** p < 0.01.

2.5. Assessment of TLR4 Protein Levels Confirms Upregulation Detected by qPCR in Scrapie-Infected Sheep

To confirm that the observed increase in TLR4 mRNA translates to changes in protein levels, TLR4 was evaluated by WB in the medulla oblongata, thalamus, hippocampus, and frontal cortex of four scrapie-infected sheep and four control sheep (Figure 9). 

In agreement with the qPCR results, the TLR4 protein levels were significantly increased (p < 0.05) in all four brain regions of the scrapie-infected sheep.

Figure 9. TLR4 protein expression in the medulla oblongata (Mo), thalamus (Th), hippocampus (Hc), and frontal cortex (Fc) of scrapie-infected sheep versus control sheep (n = 4 per group). β-actin was used as a loading control. 

The graph depicts densitometry values and indicates a significant increase in TLR4 expression in all regions in scrapie-infected versus control sheep. Data are expressed as the mean ± SEM. Mean scores were compared using the Student's t-test. * p < 0.05.

3. Discussion

In recent years, the study of the neuroinflammatory response in prion diseases has led to a better understanding of the underlying mechanisms and the consequences. 

Neuroinflammation involves reactive microgliosis and astrogliosis [43], both of which can be detected before the onset of clinical signs and precedes spongiosis and neuronal loss [7,8,18]. 

Whether microglial activation in prion diseases is beneficial or harmful remains a matter of debate. Some authors argue that microglial activation and proliferation contribute to the neuroinflammatory process and consequent neurodegeneration [8,18,44]. 

However, others point to an overall protective role of the microglia, and in vitro, studies indicate that microglia can internalize and degrade PrPSc [27,45]. In line with these findings, in vivo experiments have shown that microglial depletion decreases the survival period, probably due to reduced PrPSc clearance [46,47]. 

Therefore, it is plausible that multiple reactive microglial phenotypes exist during the disease process, exerting a neuroprotective effect in early disease stages and, as the disease progresses, giving way to a proinflammatory phenotype that elicits detrimental effects [9,10]. One hypothesis proposes that TLR signaling mediates glial cell activation in prion diseases. 

The microglia uptake of amyloid fibers in Alzheimer's disease is known to be promoted by TLR activation [48,49]. Thus, while the activation of TLRs on glial cells may play a role in the clearance of aggregated or abnormal proteins (e.g., prion protein), chronic activation may be detrimental to the host, resulting in neurotoxicity and neuronal cell death [50]. 

To date, data on the role of TLRs in prion pathogenesis are very limited. Few studies have measured changes in TLR expression in the CNS in murine models, and one has reported changes in TLR expression in the blood of lambs, with all cases using animals experimentally infected with prion disease [22,26,30].

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