Spatio-temporal expression pattern of frizzled receptors after contusive spinal cord injury in adult rats

Abstract

Background: Wnt proteins are a large family of molecules that are critically involved in multiple central nervous system (CNS) developmental processes. Experimental evidences suggest a role for this family of proteins in many CNS disorders, including spinal cord injury (SCI), which is a major neuropathology owing to its high prevalence and chronic sensorimotor functional sequelae. Interestingly, most Wnt proteins and their inhibitors are expressed in the uninjured spinal cord, and their temporal expression patterns are dramatically altered after injury. However, little is known regarding the expression of their better-known receptors, the Frizzled family, after SCI. Thus, the aim of the present study was to evaluate the expression of Frizzled receptors in the damaged spinal cord.

Findings: Based on the evidence that Wnts are expressed in the spinal cord and are transcriptionally regulated by SCI in adulthood, we analysed the spatio-temporal mRNA and protein expression patterns of Frizzled receptors after contusive SCI using quantitative RT-PCR and single and double immunohistochemistry, respectively. Our results show that almost all of the 10 known Frizzled receptors were expressed in specific spatial patterns in the uninjured spinal cords. Moreover, the Frizzled mRNAs and proteins were expressed after SCI, although their expression patterns were altered during the temporal progression of SCI. Finally, analysis of cellular Frizzled 5 expression pattern by double immunohistochemistry showed that, in the uninjured spinal cord, this receptor was expressed in neurons, oligodendrocytes, astrocytes, microglia and NG2(+) glial precursors. After injury, Frizzled 5 was not only still expressed in oligodendrocytes, astrocytes and NG2(+) glial precursors but also in axons at all evaluated time points. Moreover, Frizzled 5 was expressed in reactive microglia/macrophages from 3 to 14 days post-injury.

Conclusions: Our data suggest the involvement of Frizzled receptors in physiological spinal cord function and in the cellular and molecular events that characterise its neuropathology.

Figure 1. Temporal mRNA expression pattern of Frizzled receptors in the non-lesioned spinal cord and after SCI.

The temporal mRNA expression pattern of Fz2 (1A), Fz4 (1B), Fz5 (1C), Fz7 (1D), Fz8 (1E), Fz9 (1F) and Fz10 (1G) was quantified using quantitative RT-PCR using specific primers in the non-lesioned (NL) spinal cord and at different time points after spinal cord injury. Differences were calculated by setting the expression values of the NL samples at 1 and normalising against ribosomal 18S rRNA. In all cases, the data are presented as the mean ± SEM; * p<0.05 versus NL. hpi, hours post-injury; dpi, days post-injury; Fz, Frizzled.

Figure 2. Spatio-temporal protein expression pattern of Frizzled 2 in the non-lesioned spinal cord and after SCI.

This figure shows representative images obtained from the microscopic evaluation of sections processed by single immunohistochemistry to visualise Fz2. The analysis was performed in non-lesioned spinal cords (2A, A₁, A₂ and A₃) and lesioned spinal cords at 6 hpi (2B and B₁) and 24 hpi (2C and C₁) and at 3 dpi (2D and D₁), 7 dpi (2E, E₁ and E₂) and 14 dpi (2F, F₁ and F₂). The squares in the images showing the entire spinal cord sections correspond to the areas of higher magnification. Scale bars = 50 µm. hpi, hours post-injury; dpi, days post-injury; Fz, Frizzled.

Figure 3. Spatio-temporal protein expression pattern of Frizzled 4 in the non-lesioned spinal cord and after SCI.

This figure shows representative images obtained from the microscopic evaluation of sections processed by single immunohistochemistry to visualise Fz4. The analysis was performed in non-lesioned spinal cords (3A, A₁, A₂ and A₃) and lesioned spinal cords at 6 hpi (3B and B₁) and 24 hpi (3C and C₁) and at 3 dpi (3D and D₁), 7 dpi (3E, E₁ and E₂) and 14 dpi (3F, F₁ and F₂). The squares in the images showing the entire spinal cord sections correspond to the areas of higher magnification. Scale bars = 50 µm. hpi, hours post-injury; dpi, days post-injury; Fz, Frizzled.

Figure 4. Spatio-temporal protein expression pattern of Frizzled 5 in the non-lesioned spinal cord and after SCI.

This figure shows representative images obtained from the microscopic evaluation of sections processed by single immunohistochemistry to visualise Fz5. The analysis was performed in non-lesioned spinal cords (4A, A₁, A₂ and A₃) and lesioned spinal cords at 6 hpi (4B and B₁) and 24 hpi (4C, C₁ and C₂) and at 3 dpi (4D, D₁ and D₂), 7 dpi (4E, E₁ and E₂) and 14 dpi (4F, F₁ and F₂). The squares in the images showing the entire spinal cord sections correspond to the areas of higher magnification. Scale bars = 50 µm. hpi, hours post-injury; dpi, days post-injury; Fz, Frizzled.

Figure 5. Spatio-temporal protein expression pattern of Frizzled 7 in the non-lesioned spinal cord and after SCI.

This figure shows representative images obtained from the microscopic evaluation of sections processed by single immunohistochemistry to visualise Fz7. The analysis was performed in non-lesioned spinal cords (5A, A₁, A₂ and A₃) and lesioned spinal cords at 6 hpi (5B, B₁ and B₂) and 24 hpi (5C, C₁ and C₂) and at 3 dpi (5D, D₁ and D₂), 7 dpi (5E, E₁ and E₂) and 14 dpi (5F, F₁ and F₂). The squares in the images showing the entire spinal cord sections correspond to the areas of higher magnification. Scale bars = 50 µm. hpi, hours post-injury; dpi, days post-injury; Fz, Frizzled.

Figure 6. Spatio-temporal protein expression pattern of Frizzled 8 in the non-lesioned spinal cord and after SCI.

This figure shows representative images obtained from the microscopic evaluation of sections processed by single immunohistochemistry to visualise Fz8. The analysis was performed in non-lesioned spinal cords (6A, A₁, A₂ and A₃) and lesioned spinal cords at 6 hpi (6B and B₁) and 24 hpi (6C and C₁) and at 3 dpi (6D and D₁), 7 dpi (6E, E₁ and E₂) and 14 dpi (6F, F₁ and F₂). The squares in the images showing the entire spinal cord sections correspond to the areas of higher magnification. Scale bars = 50 µm. hpi, hours post-injury; dpi, days post-injury; Fz, Frizzled.

Figure 7. Spatio-temporal protein expression pattern of Frizzled 9 in the non-lesioned spinal cord and after SCI.

This figure shows representative images obtained from the microscopic evaluation of sections processed by single immunohistochemistry to visualise Fz9. The analysis was performed in non-lesioned spinal cords (7A, A₁, A₂ and A₃) and lesioned spinal cords at 6 hpi (7B and B₁) and 24 hpi (7C and C₁) and at 3 dpi (7D and D₁), 7 dpi (7E, E₁ and E₂) and 14 dpi (7F, F₁ and F₂). The squares in the images showing the entire spinal cord sections correspond to the areas of higher magnification. Scale bars = 50 µm. hpi, hours post-injury; dpi, days post-injury; Fz, Frizzled.

Figure 8. Spatio-temporal protein expression pattern of Frizzled 10 in the non-lesioned spinal cord and after SCI.

This figure shows representative images obtained from the microscopic evaluation of sections processed by single immunohistochemistry to visualise Fz10. The analysis was performed in non-lesioned spinal cords (8A, A₁, A₂ and A₃) and lesioned spinal cords at 6 hpi (8B and B₁) and 24 hpi (8C and C₁) and at 3 dpi (8D, D₁ and D₂), 7 dpi (8E, E₁ and E₂) and 14 dpi (8F, F₁ and F₂). The squares in the images showing the entire spinal cord sections correspond to the areas of higher magnification. Scale bars = 50 µm. hpi, hours post-injury; dpi, days post-injury; Fz, Frizzled.

Figure 9. Cellular protein expression pattern of Frizzled 5 in non-lesioned spinal cords and at 6 hpi.

This figure shows representative images obtained from the microscopic evaluation of sections processed by double immunohistochemistry to visualise Frizzled (Fz) 5 in astrocytes (glial fibrillary acidic protein (GFAP)), neurons (neuronal nuclei (NeuN)), oligodendrocytes (adenomatous polyposis coli (APC)), axons (neurofilament 200 (NF200)), microglia/macrophages (OX-42) and glial progenitors (NG2) in the non-lesioned spinal cords (9A, A₁, A₂, A₃, A₄ and A₅) and at 6 hours post-injury (hpi) (9B, B₁, B₂, B₃ and B₄). The squares in the images showing the entire spinal cord sections (9A and B) correspond to the areas of higher magnification. Scale bars = 40 µm.

Figure 10. Cellular protein expression pattern of Frizzled 5 at 24 hpi and 3 dpi.

This figure shows representative images obtained from the microscopic evaluation of sections processed by double immunohistochemistry to visualise Frizzled (Fz) 5 in astrocytes (glial fibrillary acidic protein (GFAP)), neurons (neuronal nuclei (NeuN)), oligodendrocytes (adenomatous polyposis coli (APC)), axons (neurofilament 200 (NF200)), microglia/macrophages (OX-42) and glial progenitors (NG2) at 24 hours post-injury (hpi) (10A, A₁, A₂, A₃, A₄ and A₅) and 3 days post-injury (dpi) (10B, B₁, B₂, B₃, B₄, B₅ and B₆). The squares in the images showing the entire spinal cord sections (10A and B) correspond to the areas of higher magnification. Scale bars = 40 µm.

Figure 11. Cellular protein expression pattern of Frizzled 5 at 7 and 14 dpi.

This figure shows representative images obtained from the microscopic evaluation of sections processed by double immunohistochemistry to visualise Frizzled (Fz) 5 in astrocytes (glial fibrillary acidic protein (GFAP)), neurons (neuronal nuclei (NeuN)), oligodendrocytes (adenomatous polyposis coli (APC)), axons (neurofilament 200 (NF200)), microglia/macrophages (OX-42) and glial progenitors (NG2) at 7 (11A, A₁, A₂, A₃, A₄, A₅ and A₆) and 14 days post-injury (dpi) (11B, B₁, B₂, B₃, B₄, B₅, B₆ and B₇). The squares in the images showing the entire spinal cord sections (11A and B) correspond to the areas of higher magnification. Scale bars = 40 µm.